1-(5-tert-butyl-2-phenyl-2h-pyrazol-3-yl)-3-[2-fluoro-4-(1-methyl-2-oxo-2,3-dihydro-1h-imidazo[4,5-b]pyridin-7-yloxy)-phenyl]-urea and related compounds and their use in therapy

ABSTRACT

The present invention pertains generally to the field of therapeutic compounds, and more specifically to certain compounds of the following formula (for convenience, collectively referred to herein as “IP compounds”), which, inter alia, are useful in the treatment of cancer, e.g., cancer characterised by (e.g., driven by) mutant RAS (“mutant RAS cancer”). The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions in the treatment of cancer, e.g., mutant RAS cancer.

RELATED APPLICATION

This application is related to U.S. patent application No. 61/300,085filed 1 Feb. 2010, the contents of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

The present invention pertains generally to the field of therapeuticcompounds, and more specifically to certain compounds (for convenience,collectively referred to herein as “IP compounds”), which, inter alia,are useful in the treatment of cancer, e.g., cancer characterised by(e.g., driven by) mutant RAS (“mutant RAS cancer”). The presentinvention also pertains to pharmaceutical compositions comprising suchcompounds, and the use of such compounds and compositions in thetreatment of cancer, e.g., mutant RAS cancer.

BACKGROUND

A number of patents and publications are cited herein in order to morefully describe and disclose the invention and the state of the art towhich the invention pertains. Each of these references is incorporatedherein by reference in its entirety into the present disclosure, to thesame extent as if each individual reference was specifically andindividually indicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

Ranges are often expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by the use of the antecedent “about,” itwill be understood that the particular value forms another embodiment.

This disclosure includes information that may be useful in understandingthe present invention. It is not an admission that any of theinformation provided herein is prior art or relevant to the presentlyclaimed invention, or that any publication specifically or implicitlyreferenced is prior art.

Cancer and RAS

RAS proteins are small-guanine nucleotide binding proteins that aredownstream of growth factor, cytokine and hormone receptors. These cellsurface receptors activate proteins called guanine-nucleotide exchangefactors (GNEFs), which replace GDP for GTP on RAS proteins, stimulatingRAS activation. Other proteins called GTPase-activating proteins (GAPs)stimulate the intrinsic GTPase activity of RAS, thereby promoting GTPhydrolysis and returning RAS to its inactive GDP-bound state. ActivatedRAS binds to several effector proteins, including phosphoinositide3-kinase (PI3K), the RAF family of protein kinases, and the Ralguanine-nucleotide exchange factor. These effectors in turn regulate theactivity of the signalling pathways that control cell proliferation,senescence, survival and differentiation. There are three RAS genes inmammals called HRAS, KRAS and NRAS and they serve overlapping butnon-conserved functions.

RAS proteins are also important in cancer. 20-30% of human tumoursharbour somatic gain-of-function mutations in one of the RAS genes. Mostcommonly these involve the codons for glycine 12 (G12), glycine 13 (G13)and glutamine 61 (Q61) and these mutations impair, through differentmechanisms, the GAP-stimulated intrinsic GTPase activity of RAS,trapping it in the active GTP-bound state and allowing it to promotetumorigenesis. See, e.g., Downward, 2003; Young et al., 2009; and Bos,1989.

TABLE 1 Frequency of RAS Mutatations in Different Types of CancersTumour Type Frequency Pancreas 90% Thyroid (Undifferentiated papillary)60% Thyroid (Follicular) 55% Colorectal 45% Seminoma 45% Myelodysplasticsyndrome (MDS) 40% Lung adenocarcinoma (non-small-cell) 35% Liver 30%Acute myelogenous leukemia (AML) 30% Melanoma 15% Bladder 10% Kidney 10%

RAS and BRAF

Active RAS proteins activate several downstream effectors, including theproteins of the RAF family. There are three RAF proteins, ARAF, BRAF andCRAF. Activated RAF phosphorylates and activates a second protein kinasecalled MEK, which then phosphorylates and activates a third proteinkinase called ERK. ERK phosphorylates a multitude of cytosolic andnuclear substrates, thereby regulating cell processes such asproliferation, survival, differentiation and senescence.

BRAF is important in cancer, because it is mutated in about 2% of humancancers, particularly in melanoma (43% of cases), thyroid (45%), ovarian(10%), and colorectal (13%) cancers. In contrast, ARAF and CRAFmutations are very rare in human cancer. Notably, however, in cancercells, oncogenic RAS does not signal through BRAF, but instead signalsexclusively through CRAE to activate MEK.

Over 100 different mutations have been described in BRAF in cancer, buta single mutation (a glutamic acid substitution for the valine atposition 600) accounts for about 90% of the mutations that occur. Thismutant activates BRAF 500-fold, and allows it to stimulate constitutiveERK and NEkB signalling, stimulating survival and proliferation.Consequently, ^(V600E)BRAF can transform cells such as fibroblasts andmelanocytes. Inhibition of ^(V600E)BRAF in cancer cells inhibits cellproliferation and induces apoptosis in vitro, and in vivo it suppressestumor cell growth, validating ^(V600E)BRAF as a therapeutic target.

In the vast majority of cancers, BRAF and RAS mutations are mutuallyexclusive. This provides genetic evidence to suggest that these proteinsare on the same pathway and that they drive the same processes in cancercells. However, there are clear differences between oncogenic BRAF andoncogenic RAS functions in cancer cells. First, RAS activates severalpathways, whereas BRAF is only known to activate the MEK/ERK pathway. Asa consequence, BRAF mutant cells are more dependent on MEK/ERKsignalling and so are considerably more sensitive to BRAF or MEKinhibitors than cell in which RAS is mutated. See, e.g., Garnett et al.,2004; Wellbrock et al., 2004; Gray-Schopfer et al., 2007; Solit et al.,2006.

Related Compounds

Niculescu-Duvaz et al., 2006 (WO 2006/043090 A1), describes thefollowing classes of compounds at pages 41 and 43 therein. The compoundsare described as BRAF inhibitors useful for the treatment of cancer,especially mutant BRAF cancer.

Additionally, Niculescu-Duvaz et al., 2006 provides the followingexamples:

Niculescu-Duvaz et al., 2007 (WO 2007/125330 A1), describes thefollowing classes of compounds at pages 57 and 58 therein. The compoundsare described as BRAF inhibitors useful for the treatment of cancer,especially mutant BRAF cancer.

Additionally, Niculescu-Duvaz et al., 2007 provides the followingexamples:

The present invention provides alternative compounds, which arecharacterized by a particular combination of structural motifs, andwhich provide surprising and unexpected activity (e.g., activity againstmutant RAS cancers), for example, as compared to one or more of thestructurally-related known compounds.

Although structurally-related compounds are known as BRAF inhibitors, itwould not have been predicted that the claimed compounds are activeagainst mutant RAS cancers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of several compounds of the presentinvention: AA-01, AA-02, AA-03, and AA-04.

FIG. 2 shows the chemical structure of several compounds of the presentinvention: BB-01 and BB-02.

FIG. 3 shows the chemical structure of several comparison compounds:XX-01, XX-02, XX-03, and XX-04.

FIG. 4 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for compound AA-01.The range of cell lines are (a) a panel of mutant BRAF (mutBRAF) celllines: WM266.4, A375M, UACC62; (b) a panel of mutant RAS (mutRAS) celllines: SW620, HCT116, and WM1361; and (c) a panel of wild type BRAF andRAS (wtBRAFT/RAS) cell lines: SKMEL23. KM12, and BT474.

FIG. 5 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for compound BB-01.The ranges of cell lines are as for FIG. 4.

FIG. 6 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for compound BB-02.The ranges of cell lines are as for FIG. 4.

FIG. 7 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for comparisoncompound XX-01. The ranges of cell lines are as for FIG. 4.

FIG. 8 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for comparisoncompound XX-02. The ranges of cell lines are as for FIG. 4.

FIG. 9 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for comparisoncompound XX-03. The ranges of cell lines are as for FIG. 4.

FIG. 10 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for comparisoncompound XX-04. The ranges of cell lines are as for FIG. 4.

FIG. 11 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant RAS cell line SW620, fortreatment with compound AA-01 and for controls.

FIG. 12 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant RAS cell line SW620, fortreatment with compound BB-02 and for controls.

FIG. 13 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant BRAF cell line A375, fortreatment with compound AA-01 and for controls.

FIG. 14 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant BRAF cell line A375, fortreatment with compound BB-02 and for controls.

FIG. 15 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant RAS cell line SW620, fortreatment with comparison compound XX-01 and for controls.

FIG. 16 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant RAS cell line SW620, fortreatment with comparison compound XX-02 and for controls.

FIG. 17 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant RAS cell line SW620, fortreatment with comparison compound XX-03 and for controls.

FIG. 18 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant RAS cell line SW620, fortreatment with comparison compound XX-04 and for controls.

FIG. 19 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant BRAF cell line A375, fortreatment with comparison compound XX-02 and for controls.

SUMMARY OF THE INVENTION

One aspect of the invention pertains to certain compounds (forconvenience, collectively referred to herein as “IP compounds”), asdescribed herein.

Another aspect of the invention pertains to a composition (e.g., apharmaceutical composition) comprising an IP compound, as describedherein, and a pharmaceutically acceptable carrier or diluent.

Another aspect of the invention pertains to method of preparing acomposition (e.g., a pharmaceutical composition) comprising the step ofadmixing an IP compound, as described herein, and a pharmaceuticallyacceptable carrier or diluent.

Another aspect of the present invention pertains to a method oftreatment comprising administering to a subject in need of treatment atherapeutically-effective amount of an IP compound, as described herein,preferably in the form of a pharmaceutical composition.

Another aspect of the present invention pertains to an IP compound asdescribed herein for use in a method of treatment of the human or animalbody by therapy.

Another aspect of the present invention pertains to use of an IPcompound, as described herein, in the manufacture of a medicament foruse in treatment.

In one embodiment, the treatment is treatment of cancer.

In one embodiment, the cancer is solid tumour cancer.

In one embodiment, the cancer is pancreatic cancer; thyroid (e.g.,follicular; undifferentiated papillary) cancer; colorectal cancer;seminoma; myelodysplastic syndrome (MDS); lung cancer (e.g., lungadenocarcinoma); liver cancer; leukemia (e.g., acute myelogenousleukemia (AML)); melanoma; bladder cancer; kidney cancer; breast cancer,ovarian cancer, bile duct cancer, or glioma.

In one embodiment, the cancer is pancreatic cancer.

In one embodiment, the cancer is thyroid cancer.

In one embodiment, the cancer is colorectal cancer.

In one embodiment, the cancer is seminoma.

In one embodiment, the cancer is myelodysplastic syndrome (MDS).

In one embodiment, the cancer is lung cancer.

In one embodiment, the cancer is lung adenocarcinoma.

In one embodiment, the cancer is liver cancer.

In one embodiment, the cancer is leukemia.

In one embodiment, the cancer is acute myelogenous leukemia (AML).

In one embodiment, the cancer is melanoma.

In one embodiment, the cancer is bladder cancer.

In one embodiment, the cancer is kidney cancer.

In one embodiment, the cancer is breast cancer.

In one embodiment, the cancer is ovarian cancer.

In one embodiment, the cancer is bile duct cancer.

In one embodiment, the cancer is glioma.

In one embodiment, the cancer is mutant RAS cancer (e.g., mutant RASpancreatic cancer, etc.).

In one embodiment, the cancer is characterised by, or furthercharacterised by, cancer stem cells.

In one embodiment, the treatment further comprises treatment with one ormore additional therapeutic agents or therapies, for example, one ormore additional a cancer agents or therapies.

Another aspect of the present invention pertains to a kit comprising (a)an IP compound, as described herein, preferably provided as apharmaceutical composition and in a suitable container and/or withsuitable packaging; and (b) instructions for use, for example, writteninstructions on how to administer the compound.

Another aspect of the present invention pertains to an IP compoundobtainable by a method of synthesis as described herein, or a methodcomprising a method of synthesis as described herein.

Another aspect of the present invention pertains to an IP compoundobtained by a method of synthesis as described herein, or a methodcomprising a method of synthesis as described herein.

Another aspect of the present invention pertains to novel intermediates,as described herein, which are suitable for use in the methods ofsynthesis described herein.

Another aspect of the present invention pertains to the use of suchnovel intermediates, as described herein, in the methods of synthesisdescribed herein.

As will be appreciated by one of skill in the art, features andpreferred embodiments of one aspect of the invention will also pertainto other aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION Compounds

The present relates to certain compounds which are structurally relatedto the following compounds:

In contrast to known compounds, the compounds of the present inventionare characterized by a particular combination of structural motifs,specifically:

(A) a 1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy motif:

(B) a linking 2-fluoro-phen-1,4-di-yl moiety or a linkingnaphth-1,4-di-yl motif:

(C) a 1-(5-tert-butyl-2-phenyl-2H-pyrazol-3-yl)-ureyl motif, where thephenyl group optionally bears a meta- or para-substituent:

None of he known compounds have all three of these motifs. Furthermore,comparison studies with compounds (including known compounds)demonstrate that, surprisingly and unexpectedly, compounds having allthree of the motifs (i.e., the claimed compounds) have substantiallybetter activity against mutant RAS cancers, than comparison compoundswhich lack one or more of these motifs.

Thus, one aspect of the present invention pertains to compounds of thefollowing formula, and pharmaceutically acceptable salts, hydrates, andsolvates thereof (collectively referred to herein as “IP compounds”):

wherein -J- is independently:

and wherein —R is independently —H, -Me, —F, —Cl, —Br, or —I;

and wherein —R is positioned meta- or para- on the phenyl ring.

In one embodiment, the compound is selected from compounds of thefollowing formula, and pharmaceutically acceptable salts, hydrates, andsolvates thereof, wherein —R^(A) is independently —H, —F, —Cl, —Br, or—I, and wherein —R^(A) is positioned meta- or para- on the phenyl ring:

In one embodiment, the compound is selected from compounds of thefollowing formula, and pharmaceutically acceptable salts, hydrates, andsolvates thereof, wherein —R^(A) is independently —H, —F, —Cl, —Br, or—I:

In one embodiment, the compound is selected from compounds of thefollowing formula, and pharmaceutically acceptable salts, hydrates, andsolvates thereof, wherein —R^(A) is independently —H, -Me, —F, —Cl, —Br,or —I:

In one embodiment, —R^(A) is independently —H, -Me, —F, or —Cl.

In one embodiment, —R^(A) is independently —H or -Me.

In one embodiment, —R^(A) is independently —H.

In one embodiment, —R^(A) is independently -Me.

In one embodiment, —R^(A) is independently —F or —Cl.

In one embodiment, —R^(A) is independently —F.

In one embodiment, —R^(A) is independently —Cl.

In one embodiment, the compound is selected from the following compound(i.e., AA-01), and pharmaceutically acceptable salts, hydrates, andsolvates thereof:

In one embodiment, the compound is selected from the following compound(i.e., AA-02), and pharmaceutically acceptable salts, hydrates, andsolvates thereof:

In one embodiment, the compound is selected from the following compound(i.e., AA-03), and pharmaceutically acceptable salts, hydrates, andsolvates thereof:

In one embodiment, the compound is selected from the following compound(i.e., AA-04), and pharmaceutically acceptable salts, hydrates, andsolvates thereof:

In one embodiment, the compound is selected from compounds of thefollowing formula, and pharmaceutically acceptable salts, hydrates, andsolvates thereof, wherein —R⁸ is independently —H, -Me, —F, —Cl, —Br, or—I, and wherein —R⁸ is positioned meta- or para- on the phenyl ring:

In one embodiment, the compound is selected from compounds of thefollowing formula, and pharmaceutically acceptable salts, hydrates, andsolvates thereof, wherein —R⁸ is independently —H, -Me, —F, —Cl, —Br, or—I:

and pharmaceutically acceptable salts, hydrates, and solvates thereof,wherein —R^(B) is independently —Id, -Me, —F, —Cl, —Br, or —I:

In one embodiment, —R^(B) is independently —H, -Me, —F, or —Cl.

In one embodiment, —R^(B) is independently —H or -Me.

In one embodiment, —R^(B) is independently —H.

In one embodiment, —R^(B) is independently -Me.

In one embodiment, —R^(B) is independently —F or —Cl.

In one embodiment, —R^(B) is independently —F.

In one embodiment, —R^(E)' is independently —Cl.

In one embodiment, the compound is selected from the following compound(i.e., BB-01), and pharmaceutically acceptable salts, hydrates, andsolvates thereof:

In one embodiment, the compound is selected from the following compound(i.e., BB-02), and pharmaceutically acceptable salts, hydrates, andsolvates thereof:

Combinations

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the chemical groups represented by the variables (e.g.,-J-, —R, —R^(A), —R^(B), etc.) are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed, to the extent that suchcombinations embrace compounds that are stable compounds (i.e.,compounds that can be isolated, characterised, and tested for biologicalactivity). In addition, all sub-combinations of the chemical groupslisted in the embodiments describing such variables are alsospecifically embraced by the present invention and are disclosed hereinjust as it each and every such sub-combination of chemical groups wasindividually and explicitly disclosed herein.

Substantially Purified Forms

One aspect of the present invention pertains to IP compounds, asdescribed herein, in substantially purified form and/or in a formsubstantially free from contaminants.

In one embodiment, the compound is in substantially purified form and/orin a form substantially free from contaminants.

In one embodiment, the compound is in a substantially purified form witha purity of least 50% by weight, e.g., at least 60% by weight, e.g., atleast 70% by weight, e.g., at least 80% by weight, e.g., at least 90% byweight, e.g., at least 95% by weight, e.g., at least 97% by weight,e.g., at least 98% by weight, e.g., at least 99% by weight.

Unless specified, the substantially purified form refers to the compoundin any stereoisomeric form. For example, in one embodiment, thesubstantially purified form refers to a mixture of stereoisomers, i.e.,purified with respect to other compounds. In one embodiment, thesubstantially purified form refers to one stereoisomer.

In one embodiment, the compound is in a form substantially free fromcontaminants wherein the contaminants represent no more than 50% byweight, e.g., no more than 40% by weight, e.g., no more than 30% byweight, e.g., no more than 20% by weight, e.g., no more than 10% byweight, e.g., no more than 5% by weight, e.g., no more than 3% byweight, e.g., no more than 2% by weight, e.g., no more than 1% byweight.

Unless specified, the contaminants refer to other compounds, that is,other than stereoisomers. In one embodiment, the contaminants refer toother compounds and other stereoisomers.

Isomers

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R—, S—, and meso-forms; D- and L-forms; d-and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

Note that specifically excluded from the term “isomers,” as used herein,are structural (or constitutional) isomers (i.e., isomers which differin the connections between atoms rather than merely by the position ofatoms in space). For example, a reference to a tert-butyl group,—C(CH₃)₃, is not to be construed as a reference to its structuralisomer, iso-butyl, —CH₂CH(CH₃)₂. Similarly, a reference topara-chlorophenyl is not to be construed as a reference to itsstructural isomer, meta-chlorophenyl.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²11 (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including mixtures thereof.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19.

For example, if the compound is cationic, or has a functional groupwhich may be cationic (e.g., —NH— may be —NH₂ ⁺—), then a salt may beformed with a suitable anion. Examples of suitable inorganic anionsinclude, but are not limited to, those derived from the followinginorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric,sulfurous, nitric, nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examplesof suitable polymeric organic anions include, but are not limited to,those derived from the following polymeric acids: tannic acid,carboxymethyl cellulose.

Unless otherwise specified, a reference to a particular compound alsoincludes salt forms thereof.

Hydrates and Solvates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the compound. The term “solvate” is used hereinin the conventional sense to refer to a complex of solute (e.g.,compound, salt of compound) and solvent. If the solvent is water, thesolvate may be conveniently referred to as a hydrate, for example, amono-hydrate, a di-hydrate, a tri-hydrate, etc.

Unless otherwise specified, a reference to a particular compound alsoincludes solvate and hydrate forms thereof.

Chemical Synthesis

Methods for the chemical synthesis of compounds of the present inventionare described herein. These and/or other well known methods may bemodified and/or adapted in known ways in order to facilitate thesynthesis of additional compounds within the scope of the presentinvention.

Descriptions of general laboratory methods and procedures, useful forthe preparation of the compounds described herein, are provided inVogel's Textbook of Practical Organic Chemistry 5th Edition, 1989,(Editors: Furniss, Hannaford, Smith, and Tatchell) (published byLongmann, UK).

Methods for the synthesis of pyridine compounds in particular aredescribed in Heterocyclic Chemistry, 3rd Edition, 1998, (Editors: Joule,Mills, and Smith) (published by Chapman & Hall, UK).

The IP compounds described herein may be prepared via intermediates (2).These intermediates may be prepared from commercially available startingmaterial, 2-amino-3-nitro-4-chloropyridine (1), and3-fluoro-4-aminophenol (R¹ is —H and R² is —F) or 4-amino-1-naphthol (R¹and R₂ together are —CH═CH—). Intermediates (2) are then protectedselectively at the amino group, for example as a BOC carbamate, toafford intermediates (3).

The intermediates (3) can also be obtained directly from2-amino-3-nitro-4-chloropyridine (1) and N-BOC-protected3-fluoro-4-aminophenol or N-BOC-protected 4-amino-1-naphthol.

The nitro group of the protected intermediates (3) may be reduced to anamino group with Pd/C and ammonium formate or hydrogen, or with NiCl₂and NaBH₄, to give the diamino intermediates (4).

The intermediates (8), alkylated at N3 (by respect to the pyridinering), may be prepared from intermediates (4). The more nucleophilic3-amino group on intermediates (4) is converted to ethyl carbamate, toafford intermediates (5), and the BOC group is removed with TFA toafford intermediates (6). Deprotonation of the acidic carbamate protonwith NaH gives an anion on N3 that is alkylated to afford theintermediates (7). Cyclisation of intermediates (7), in the presence ofbase, gives the corresponding intermediates (8).

The intermediates (8) are reacted with3-tert-butyl-5-isocyanato-1-aryl-1H-pyrazoles to afford thecorresponding ureas. The respective isocyanates can be obtained eitherby the reaction of amines with phosgene, triphosgene or theirderivatives, or by conversion of the corresponding carboxylic acids toacyl azides with, for example, diphenyl phosphoryl azide, followed byCurtius rearrangement. The aryl group on the pyrazole may be, forexample, unsubstituted or substituted (e.g., meta or para-substituted)with an alkyl group (e.g., -Me) or a halogen atom (e.g., —F, —Cl, —Br,—I).

Compositions

One aspect of the present invention pertains to a composition (e.g., apharmaceutical composition) comprising an IP compound, as describedherein, and a pharmaceutically acceptable carrier, diluent, orexcipient.

In one embodiment, the composition further comprises one or more (e.g.,1, 2, 3, 4) additional therapeutic agents, as described herein.

Another aspect of the present invention pertains to a method ofpreparing a composition (e.g., a pharmaceutical composition) comprisingadmixing an IP compound, as described herein, and a pharmaceuticallyacceptable carrier, diluent, or excipient.

Another aspect of the present invention pertains to a method ofpreparing a composition (e.g., a pharmaceutical composition) comprisingadmixing an IP compound, as described herein; one or more (e.g., 1, 2,3, 4) additional therapeutic agents, as described herein; and apharmaceutically acceptable carrier, diluent, or excipient.

Uses

The compounds described herein are useful, for example, in the treatmentof cancer, for example, mutant RAS cancer.

For the avoidance of doubt, the term “mutant RAS cancer” is used hereino refer to cancer that is characterised by (e.g., driven by) mutant RAS,for example, by one or more mutations (e.g., gain-of-function mutations)in one of the RAS genes (i.e., HRAS, KRAS, and NRAS). As discussedherein, the most common RAS mutations involve codons for one or more ofglycine 12 (G12), glycine 13 (G13) and glutamine 61 (061).

Use in Methods of Therapy

Another aspect of the present invention pertains to an IP compound, asdescribed herein, for use in a method of treatment of the human oranimal body by therapy.

Another aspect of the present invention pertains to an IP compound, asdescribed herein, in combination with one or more (e.g., 1, 2, 3, 4)additional therapeutic agents, as described herein, for use in a methodof treatment of the human or animal body by therapy.

Use in the Manufacture of Medicaments

Another aspect of the present invention pertains to use of an IPcompound, as described herein, in the manufacture of a medicament foruse in treatment.

In one embodiment, the medicament comprises the IP compound.

Another aspect of the present invention pertains to use of an IPcompound, as described herein, and one or more (e.g., 1, 2, 3, 4)additional therapeutic agents, as described herein, in the manufactureof a medicament for use in treatment.

In one embodiment, the medicament comprises the IP compound and the oneor more (e.g., 1, 2, 3, 4) additional therapeutic agents.

Methods of Treatment

Another aspect of the present invention pertains to a method oftreatment comprising administering to a patient in need of treatment atherapeutically effective amount of an IP compound, as described herein,preferably in the form of a pharmaceutical composition.

Another aspect of the present invention pertains to a method oftreatment comprising administering to a patient in need of treatment atherapeutically effective amount of an IP compound, as described herein,preferably in the form of a pharmaceutical composition, and one or more(e.g., 1, 2, 3, 4) additional therapeutic agents, as described herein,preferably in the form of a pharmaceutical composition.

Conditions Treated

In one embodiment, the treatment is treatment of cancer.

In one embodiment, the cancer is lung cancer, small cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, stomach cancer,bowel cancer, colorectal cancer, thyroid cancer, breast cancer, ovariancancer, endometrial cancer, prostate cancer, testicular cancer, livercancer, kidney cancer, renal cell carcinoma, bladder cancer, pancreaticcancer, brain cancer, glioma, sarcoma, osteosarcoma, bone cancer,nasopharyngeal cancer (e.g., head cancer, neck cancer), skin cancer,squamous cancer, Kaposi's sarcoma, melanoma, malignant melanoma,lymphoma, or leukemia.

In one embodiment, the cancer is:

-   -   a carcinoma, for example a carcinoma of the bladder, breast,        colon/rectum (e.g., colorectal carcinomas such as colon        adenocarcinoma and colon adenoma), kidney, epidermal, liver,        lung (e.g., adenocarcinoma, small cell lung cancer and non-small        cell lung carcinomas), oesophagus, gall bladder, ovary, pancreas        (e.g., exocrine pancreatic carcinoma), stomach, cervix, thyroid,        prostate, skin (e.g., squamous cell carcinoma);    -   a hematopoietic tumour of lymphoid lineage, for example        leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell        lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell        lymphoma, or Burkett's lymphoma;    -   a hematopoietic tumor of myeloid lineage, for example acute and        chronic myelogenous leukemias, myelodysplastic syndrome, or        promyelocytic leukemia;    -   a tumour of mesenchymal origin, for example fibrosarcoma or        habdomyosarcoma;    -   a tumor of the central or peripheral nervous system, for example        astrocytoma, neuroblastoma, glioma or schwannoma;    -   melanoma; seminoma; teratocarcinoma; osteosarcoma; xenoderoma        pigmentoum; keratoctanthoma; thyroid follicular cancer; or        Kaposi's sarcoma.

In one embodiment, the cancer is solid tumour cancer.

In one embodiment, the cancer is pancreatic cancer; thyroid (e.g.,follicular; undifferentiated papillary) cancer; colorectal cancer;seminoma; myelodysplastic syndrome (MDS); lung cancer (e.g., lungadenocarcinoma); liver cancer; leukemia (e.g., acute myelogenousleukemia (AML)); melanoma; bladder cancer; kidney cancer; breast cancer,ovarian cancer, bile duct cancer, or glioma.

In one embodiment, the cancer is pancreatic cancer.

In one embodiment, the cancer is thyroid cancer.

In one embodiment, the cancer is colorectal cancer.

In one embodiment, the cancer is seminoma.

In one embodiment, the cancer is myelodysplastic syndrome (MDS).

In one embodiment, the cancer is lung cancer.

In one embodiment, the cancer is lung adenocarcinoma.

In one embodiment, the cancer is liver cancer.

In one embodiment, the cancer is leukemia.

In one embodiment, the cancer is acute myelogenous leukemia (AML).

In one embodiment, the cancer is melanoma.

In one embodiment, the cancer is bladder cancer.

In one embodiment, the cancer is kidney cancer.

In one embodiment, the cancer is breast cancer.

In one embodiment, the cancer is ovarian cancer.

In one embodiment, the cancer is bile duct cancer.

In one embodiment, the cancer is glioma.

In one embodiment, the cancer is mutant RAS cancer (e.g., mutant RASpancreatic cancer, etc.).

In one embodiment, the cancer is characterised by, or furthercharacterised by, cancer stem cells.

The anti-cancer effect may arise through one or more mechanisms,including but not limited to, the regulation of cell proliferation, theinhibition of cell cycle progression, the inhibition of angiogenesis(the formation of new blood vessels), the inhibition of metastasis (thespread of a tumour from its origin), the inhibition of cell migration(the spread of cance cells to other parts of the body), the inhibitionof invasion (the spread of tumour cells into neighbouring normalstructures), or the promotion of apoptosis (programmed cell death). Thecompounds of the present invention may be used in the treatment of thecancers described herein, independent of the mechanisms discussedherein.

Treatment

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, alleviatiation of symptoms ofthe condition, amelioration of the condition, and cure of the condition.Treatment as a prophylactic measure (i.e., prophylaxis) is alsoincluded. For example, use with patients who have not yet developed thecondition, but who are at risk of developing the condition, isencompassed by the term “treatment.”

For example, treatment includes the prophylaxis of cancer, reducing theincidence of cancer, reducing the severity of cancer, alleviating thesymptoms of cancer, etc.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of a compound, or a material, composition or dosage formcomprising a compound, which is effective for producing some desiredtherapeutic effect, commensurate with a reasonable benefit/risk ratio,when administered in accordance with a desired treatment regimen.

Combination Therapies

The term “treatment” includes combination treatments and therapies, inwhich two or more treatments or therapies are combined, for example,sequentially or simultaneously. For example, the compounds describedherein may also be used in combination therapies, e.g., in conjunctionwith other agents, for example, cytotoxic agents, anticancer agents,etc. Examples of treatments and therapies include, but are not limitedto, chemotherapy (the administration of active agents, including, e.g.,drugs, antibodies (e.g., as in immunotherapy), prodrugs (e.g., as inphotodynamic therapy, GDEPT, ADEPT, etc.); surgery; radiation therapy;photodynamic therapy; gene therapy; and controlled diets.

For example, it may be beneficial to combine treatment with a compoundas described herein with one or more other (e.g., 1, 2, 3, 4) agents ortherapies that regulates cell growth or survival or differentiation viaa different mechanism, thus treating several characteristic features ofcancer development.

One aspect of the present invention pertains to a compound as describedherein, in combination with one or more additional therapeutic agents,as described below.

The particular combination would be at the discretion of the physicianwho would select dosages using his common general knowledge and dosingregimens known to a skilled practitioner.

The agents (i.e., the compound described herein, plus one or more otheragents) may be administered simultaneously or sequentially, and may beadministered in individually varying dose schedules and via differentroutes. For example, when administered sequentially, the agents can beadministered at closely spaced intervals (e.g., over a period of 5-10minutes) or at longer intervals (e.g., 1, 2, 3, 4 or more hours apart,or even longer periods apart where required), the precise dosage regimenbeing commensurate with the properties of the therapeutic agent(s).

The agents (i.e., the compound described here, plus one or more otheragents) may be formulated together in a single dosage form, oralternatively, the individual agents may be formulated separately andpresented together in the form of a kit, optionally with instructionsfor their use.

Other Uses

The IP compounds described herein may also be used as part of an invitro assay, for example, in order to determine whether a candidate hostis likely to benefit from treatment with the compound in question.

The IP compounds described herein may also be used as a standard, forexample, in an assay, in order to identify other compounds, otheranti-cancer agents, etc.

Kits

One aspect of the invention pertains to a kit comprising (a) an IPcompound as described herein, or a composition comprising an IP compoundas described herein, e.g., preferably provided in a suitable containerand/or with suitable packaging; and (b) instructions for use, e.g.,written instructions on how to administer the compound or composition.

In one embodiment, the kit further comprises one or more (e.g., 1, 2, 3,4) additional therapeutic agents, as described herein.

The written instructions may also include a list of indications forwhich the active ingredient is a suitable treatment.

Route Administration

The IP compound or pharmaceutical composition comprising the IP compoundmay be administered to a subject by ally convenient route ofadministration, whether systemically/peripherally or topically (i.e., atthe site of desired action).

Routes of administration include, but are not limited to, oral (e.g., byingestion); buccal; sublingual; transdermal (including, e.g., by apatch, plaster, etc.); transmucosal (including, e.g., by a patch,plaster, etc.); intranasal (e.g., by nasal spray, drops or from anatomiser or dry powder delivery device); ocular (e.g., by eyedrops);pulmonary (e.g., by inhalation or insufflation therapy using, e.g., anaerosol, e.g., through the mouth or nose); rectal (e.g., by suppositoryor enema); vaginal (e.g., by pessary); parenteral, for example, byinjection, including subcutaneous, intradermal, intramuscular,intravenous, intraarterial, intracardiac, intrathecal, intraspinal,intracapsular, subcapsular, intraorbital, intraperitoneal,intratracheal, subcuticular, intraarticular, subarachnoid, andintrasternal; by implant of a depot or reservoir, for example,subcutaneously or intramuscularly.

The Subject/Patient

The subject/patient may be a chordate, a vertebrate, a mammal, aplacental mammal, a marsupial (e.g., kangaroo, wombat), a rodent (e.g.,a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), alagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog),feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig),ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., amonkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g.,gorilla, chimpanzee, orangutang, gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development,for example, a foetus.

In one preferred embodiment, the subject/patient is a human.

Formulations

While it is possible for the IP compound to be administered alone, it ispreferable to present it as a pharmaceutical formulation (e.g.,composition, preparation, medicament) comprising at least one IPcompound, as described herein, together with one or more otherpharmaceutically acceptable ingredients well known to those skilled inthe art, including, but not limited to, pharmaceutically acceptablecarriers, diluents, excipients, adjuvants, fillers, buffers,preservatives, anti-oxidants, lubricants, stabilisers, solubilisers,surfactants (e.g., wetting agents), masking agents, colouring agents,flavouring agents, and sweetening agents. The formulation may furthercomprise other active agents, for example, other therapeutic orprophylactic agents.

Thus, the present invention further provides pharmaceuticalcompositions, as defined above, and methods of making a pharmaceuticalcomposition comprising admixing at least one IP compound, as describedherein, together with one or more other pharmaceutically acceptableingredients well known to those skilled in the art, e.g., carriers,diluents, excipients, etc. If formulated as discrete units (e.g.,tablets, etc.), each unit contains a predetermined amount (dosage) ofthe compound.

The term “pharmaceutically acceptable,” as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbookof Pharmaceutical Excipients, 5th edition, 2005.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association thecompound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the compound with carriers (e.g.,liquid carriers, finely divided solid carrier, etc.), and then shapingthe product, if necessary.

The formulation may be prepared to provide for rapid or slow release;immediate, delayed, timed, or sustained release; or a combinationthereof.

Formulations may suitably be in the form of liquids, solutions (e.g.,aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous),emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups,electuaries, mouthwashes, drops, tablets (including, e.g., coatedtablets), granules, powders, losenges, pastilles, capsules (including,e.g., hard and soft gelatin capsules), cachets, pills, ampoules,boluses, suppositories, pessaries, tinctures, gels, pastes, ointments,creams, lotions, oils, foams, sprays, mists, or aerosols.

Formulations may suitably be provided as a patch, adhesive plaster,bandage, dressing, or the like which is impregnated with one or morecompounds and optionally one or more other pharmaceutically acceptableingredients, including, for example, penetration, permeation, andabsorption enhancers. Formulations may also suitably be provided in theform of a depot or reservoir.

The compound may be dissolved in, suspended in, or admixed with one ormore other pharmaceutically acceptable ingredients. The compound may bepresented in a liposome or other microparticulate which is designed totarget the compound, for example, to blood components or one or moreorgans.

Formulations suitable for oral administration (e.g., by ingestion)include liquids, solutions (e.g., aqueous, non-aqueous), suspensions(e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water,water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders,capsules, cachets, pills, ampoules, boluses.

Formulations suitable for buccal administration include mouthwashes,losenges, pastilles, as well as patches, adhesive plasters, depots, andreservoirs. Losenges typically comprise the compound in a flavoredbasis, usually sucrose and acacia or tragacanth. Pastilles typicallycomprise the compound in an inert matrix, such as gelatin and glycerin,or sucrose and acacia. Mouthwashes typically comprise the compound in asuitable liquid carrier.

Formulations suitable for sublingual administration include tablets,losenges, pastilles, capsules, and pills.

Formulations suitable for oral transmucosal administration includeliquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g.,aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil),mouthwashes, losenges, pastilles, as well as patches, adhesive plasters,depots, and reservoirs.

Formulations suitable for non-oral transmucosal administration includeliquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g.,aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil),suppositories, pessaries, gels, pastes, ointments, creams, lotions,oils, as well as patches, adhesive plasters, depots, and reservoirs.

Formulations suitable for transdermal administration include gels,pastes, ointments, creams, lotions, and oils, as well as patches,adhesive plasters, bandages, dressings, depots, and reservoirs.

Tablets may be made by conventional means, e.g., compression ormoulding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing in a suitable machine thecompound in a free-flowing form such as a powder or granules, optionallymixed with one or more binders (e.g., povidone, gelatin, acacia,sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers ordiluents (e.g., lactose, microcrystalline cellulose, calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, silica);disintegrants (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose); surface-active ordispersing or wetting agents (e.g., sodium lauryl sulfate);preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,sorbic acid); flavours, flavour enhancing agents, and sweeteners.Moulded tablets may be made by moulding in a suitable machine a mixtureof the powdered compound moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and may be formulated so asto provide slow or controlled release of the compound therein using, forexample, hydroxypropylmethyl cellulose in varying proportions to providethe desired release profile. Tablets may optionally be provided with acoating, for example, to affect release, for example an enteric coating,to provide release in parts of the gut other than the stomach.

Ointments are typically prepared from the compound and a paraffinic or awater-miscible ointment base.

Creams are typically prepared from the compound and an oil-in-watercream base. If desired, the aqueous phase of the cream base may include,for example, at least about 30% w/w of a polyhydric alcohol, i.e., analcohol having two or more hydroxyl groups such as propylene glycol,butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycoland mixtures thereof. The topical formulations may desirably include acompound which enhances absorption or penetration of the compoundthrough the skin or other affected areas. Examples of such dermalpenetration enhancers include dimethylsulfoxide and related analogues.

Emulsions are typically prepared from the compound and an oily phase,which may optionally comprise merely an emulsifier (otherwise known asan emulgent), or it may comprises a mixture of at least one emulsifierwith a fat or an oil or with both a fat and an oil. Preferably, ahydrophilic emulsifier is included together with a lipophilic emulsifierwhich acts as a stabiliser. It is also preferred to include both an oiland a fat. Together, the emulsifier(S) with or without stabiliser(s)make up the so-called emulsifying wax, and the wax together with the oiland/or fat make up the so-called emulsifying ointment base which formsthe oily dispersed phase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodiumlauryl sulfate. The choice of suitable oils or fats for the formulationis based on achieving the desired cosmetic properties, since thesolubility of the compound in most oils likely to be used inpharmaceutical emulsion formulations may be very low. Thus the creamshould preferably be a non-greasy, non-staining and washable productwith suitable consistency to avoid leakage from tubes or othercontainers. Straight or branched chain, mono- or dibasic alkyl esterssuch as di-isoadipate, isocetyl stearate, propylene glycol diester ofcoconut fatty acids, isopropyl myristate, decyl oleate, isopropylpalmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branchedchain esters known as Crodamol CAP may be used, the last three beingpreferred esters. These may be used alone or in combination depending onthe properties required. Alternatively, high melting point lipids suchas white soft paraffin and/or liquid paraffin or other mineral oils canbe used.

Formulations suitable for intranasal administration, where the carrieris a liquid, include, for example, nasal spray, nasal drops, or byaerosol administration by nebuliser, include aqueous or oily solutionsof the compound.

Formulations suitable for intranasal administration, where the carrieris a solid, include, for example, those presented as a coarse powderhaving a particle size, for example, in the range of about 20 to about500 microns which is administered in the manner in which snuff is taken,i.e., by rapid inhalation through the nasal passage from a container ofthe powder held close up to the nose.

Formulations suitable for pulmonary administration (e.g., by inhalationor insufflation therapy) include those presented as an aerosol sprayfrom a pressurised pack, with the use of a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.

Formulations suitable for ocular administration include eye dropswherein the compound is dissolved or suspended in a suitable carrier,especially an aqueous solvent for the compound.

Formulations suitable for rectal administration may be presented as asuppository with a suitable base comprising, for example, natural orhardened oils, waxes, fats, semi-liquid or liquid polyols, for example,cocoa butter or a salicylate; or as a solution or suspension fortreatment by enema.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition o he compound, such carriers as are known in theart to be appropriate.

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the compound isdissolved, suspended, or otherwise provided (e.g., in a liposome orother microparticulate). Such liquids may additional contain otherpharmaceutically acceptable ingredients, such as anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, suspending agents, thickeningagents, and solutes which render the formulation isotonic with the blood(or other relevant bodily fluid) of the intended recipient. Examples ofexcipients include, for example, water, alcohols, polyols, glycerol,vegetable oils, and the like. Examples of suitable isotonic carriers foruse in such formulations include Sodium Chloride Injection, RingersSolution, or Lactated Ringer's Injection. Typically, the concentrationof the compound in the liquid is from about 1 ng/mL to about 10 μg/mL,for example from about 10 ng/mL to about 1 μg/mL. The formulations maybe presented in unit-dose or multi-dose sealed containers, for example,ampoules and vials, and may be stored in a freeze-dried (lyophilised)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the IP compounds, and compositions comprising the IPcompounds, can vary from patient to patient. Determining the optimaldosage will generally involve the balancing of the level of therapeuticbenefit against any risk or deleterious side effects. The selecteddosage level will depend on a variety of factors including, but notlimited to, the activity of the particular IP compound, the route ofadministration, the time of administration, the rate of excretion of theIP compound, the duration of the treatment, other drugs, compounds,and/or materials used in combination, the severity of the condition, andthe species, sex, age, weight, condition, general health, and priormedical history of the patient. The amount of IP compound and route ofadministration will ultimately be at the discretion of the physician,veterinarian, or clinician, although generally the dosage will beselected to achieve local concentrations at the site of action whichachieve the desired effect without causing substantial harmful ordeleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the IP compound is in the range of about10 μg to about 250 mg (more typically about 100 μg to about 25 mg) perkilogram body weight of the subject per day. Where the compound is asalt, an ester, an amide, a prodrug, or the like, the amountadministered is calculated on the basis of the parent compound and sothe actual weight to be used is increased proportionately.

EXAMPLES

The following examples are provided solely to illustrate the presentinvention and are not intended to limit the scope of the invention, asdescribed herein.

Chemical Synthesis

All starting materials, reagents and solvents for reactions were reagentgrade and used as purchased. Chromatography solvents were HPLC grade andwere used without further purification. Reactions were monitored by thinlayer chromatography (TLC) analysis using Merck silica gel 60 F-254 thinlayer plates. Flash column chromatography was carried out on Mercksilica gel 60 (0.015-0.040 mm) or in disposable !solute Flash Si and SiII silica gel columns. Preparative TLC was performed on eitherMacherey-Nagel [809 023] pre-coated TLC plates SIL G-25 UV₂₅₄ orAnaltech [2015] pre-coated preparative TLC plates, 2000 μm with L/₂₅₄.LCMS analyses were performed on a Micromass LCT/Water's Alliance 2795HPLC system with a Discovery 5 μm, C18, 50 mm×4.6 mm i.d. column fromSupelco at a temperature of 22° C. using the following solvent systems:Solvent A: Methanol; Solvent B: 0.1% formic acid in water at a flow rateof 1 mL/min. Gradient starting with 10% A/90% B from 0-0.5 minutes then10% A/90% B to 90% A/10% B from 0.5 minutes to 6.5 minutes andcontinuing at 90% A/10% B up to 10 minutes. From 10-10.5 minutes thegradient reverted back to 10% A/90% where the concentrations remaineduntil 12 minutes. UV detection was at 254 nm and ionisation was positiveor negative ion electrospray. Molecular weight scan range is 50-1000.Samples were supplied as 1 mg/mL in DMSO or methanol with 3 μL injectedon a partial loop fill. NMR spectra were recorded in DMSO-d₆ on a BrukerAdvance 500 MHz spectrometer.

Synthesis 1 Tert-butyl 2-fluoro-4-hydroxyphenylcarbamate

4-Amino-3-fluorophenol (10.61 g, 83.5 mmol) was added to a moltenmixture of Boc₂O (18.29 g, 83.8 mmol) and InCl₃ (188 mg, 0.85 mmol) at35° C. The black mixture was stirred at 35° C. for 2 hours, during whichtime it turned into a thick black oil. The mixture was then diluted withEtOAc (200 mL) and H₂O (200 mL) and stirring was continued for 10minutes. The layers were separated and the organic layer was washed withH₂O (3×200 mL), dried (MgSO₄), filtered and concentrated to dryness. Theresulting black oil was redissolved in CH₂Cl₂ (50 mL) and loaded onto asilica gel column. Elution with 5→7% EtOAc in CH₂Cl₂ furnished the titlecompound as a light yellow, crystalline solid.

Yield: 16.7 g (90%). ¹H-NMR (DMSO-d₆), δ (ppm), J (Hz): 1.42 (s, 9H,C(CH₃)₃), 6.50-6.57 (m, 2H, ArH), 7.11-7.21 (m, 1H, ArH), 8.45 (bs, 1H,OH), 9.63 (s, 1H, NHBoc); ¹³C-NMR (DMSO-d₆), δ (ppm), J (Hz): 28.0,78.6, 1027(d. J_(FH)=22.2), 110.8 (d, J_(FH)=2.7), 117.1 (d,J_(FH)=12.6), 127.2, 153.7, 155.5 (d, J_(FH)=11.3), 156.1 (d,J_(FH)=246); ¹⁹F-NMR (DMSO-d₆), δ (ppm): −121.6; LC-MS (3.94 min): m/zcalcd. for C₁₁H₁₄FNO₃ [M-C(CH₃)₃]⁺: 172.0; found: 172.0.

Synthesis 2 Tert-butyl4-(2-amino-3-nitropyridin-4-yloxy)-2-fluorophenylcarbamate (3a)

Dry DMSO (20 mL) was added to NaH (1.029 g of a 60% dispersion inmineral oil, 25.7 mmol) in a round bottom flask under argon. After 5minutes, solid tert-butyl 2-fluoro-4-hydroxyphenylcarbamate (5.59 g,24.6 mmol) was added in three portions, giving a dark solution, which,after 15 minutes of stirring at room temperature, was treated with4-chloro-3-nitropyridin-2-amine (4.23 g, 24.4 mmol) at once. The darkred solution was heated to 110° C. for 1 hour and allowed to cool downto room temperature. EtOAc (150 mL) and H₂O (200 mL) were subsequentlyadded to the solution and the organic layer was isolated. The aqueouslayer was extracted with EtOAc (3×100 mL) and the combined organiclayers were washed once with saturated NaHCO₃ (150 mL), dried (MgSO₄),filtered, and concentrated to dryness to give a bright yellow solid.This material was used in the next step without further purification.

Yield: 8.68 g (98%). ¹H-NMR (DMSO-d₆), δ (ppm), J (Hz): 1.46 (s, 9H,C(CH₃)₃), 6.08 (d, 1H, ³J_(HH)=5.5, PyrH), 7.01 (m, 1H, ArH), 7.18 (brs, 2H, NH₂), 7.22 (m, 1H, ArH), 7.67 (m, 1H, ArH), 8.04 (d, 1H,³J_(HH)=5.5, PyrH), 9.03 (s, 1H, NHBoc); ¹⁹F-NMR (DMSO-d₆), δ (ppm):−120.7; LC-MS (4.72 min): m/z calcd. for C₁₆H₁₇FN₄O₆ [M+H⁺]: 365.0;found: 365.0.

Synthesis 3 Tert-butyl4-(2,3-diaminopyridin-4-yloxy)-2-fluorophenylcarbamate (4a)

Pd/C (1.09 g) was added to a yellow solution of tert-butyl4-(2-amino-3-nitropyridin-4-yloxy)-2-fluorophenylcarbamate (3a) (6.20 g,17.0 mmol) in EtOAc/EtOH (90/150 mL) and the black mixture was stirredunder a nitrogen atmosphere for 5 hours and filtered over Celite. Thedark brown filtrate was concentrated to dryness, redissolved in CH₂Cl₂(20 mL) and loaded onto a silica gel column. The products were elutedwith EtOAc and the fractions containing the title compound wereevaporated to dryness. The orange oil was dissolved in CH₂Cl₂ and anequal amount of hexane was added. The solution was concentrated todryness to give an orange foam.

Yield: 4.30 g (76%). ¹H-NMR (DMSO-d₆), δ (ppm), J (Hz): 1.45 (s, 9H,C(CH₃)₃), 4.47 (s, 2H, NH₂), 5.61 (s, 2H, NH₂), 6.09 (d, 1H,³J_(HH)=5.5, PyrH), 6.76 (m, 1H, ArH), 6.87 (m, 1H, ArH), 7.28 (d, 1H,³J_(HH)=5.5, PyrH), 7.47 (m, 1H, ArH), 8.82 (s, 1H, NHBoc); ¹³C-NMR(DMSO-d₆), δ (ppm), J (Hz): 28.0, 79.1, 104.4, 105.9 (d, J_(FH)=23.1),113.4 (d, J_(FH)=3.1), 120.3, 121.5 (d, J_(FH)=12.2), 126.1, 135.7,146.2, 150.5, 153.1 (d, J_(FH)=10.1), 153.3, 155.1 (d, J_(FH)=248);¹⁹F-NMR (DMSO-d₆), δ (ppm): −120.7; LC-MS (2.69 min): m/z calcd. forC₁₆H₂₀FN₄O₃ [M+H⁺]: 335.2; found: 335.3.

Synthesis 4 Ethyl4-(4-N-(tert-butoxycarbonyl)-amino-3-fluorophenoxy)-2-aminopyridin-3-yl-carbamate(5a)

4-(4-N-(tert-butoxycarbonyl)-amino-3-fluorophenyloxy)-2,3-diaminopyridine(4a) (2.5 g, 7.5 mmol) was dissolved in dry THF (50 mL) under stirring,pyridine (1.2 mL, 15 mmol) was added and the solution was cooled at 0°C. Ethyl chloroformate (0.77 mL, 8.0 mmol) was added at once. After 30minutes, the reaction mixture was allowed to reach room temperature andstirred for further 24 hours. The solvent was evaporated under vacuumand the residue partitioned between DCM and saturated aqueous Na₂CO₃.The organic layer was washed with H₂O, dried over MgSO₄ and evaporated.The residue was purified by column chromatography (eluent gradient DCMto EtOAc) to afford the title compound as a foam.

Yield: 1.13 g, 37%. ¹H-NMR δ: 1.17 (t, 3H, CH_(3,Et), J=7.1 Hz), 1.45(s, 9H, tBu), 4.02 (q, 2H, J=7.1, CH_(2,Et)), 5.89 (s, 2H, NH_(2,Py2)),5.98 (d, 1H, J=5.7, H_(Py)), 6.83 (d, 1H, H_(arom)), 6.96 (d, 1H,H_(arom)), 7.55 (t, 1H, H_(arom)), 7.73 (d, 1H, J=5.7, H_(Py)), 8.29 (brs, 1H, NH_(Py3)), 9.39 (s, 1H, NHBoc).

Synthesis 5 Ethyl4-(4-amino-3-fluorophenoxy)-2-aminopyridin-3-yl-carbamate (6a)

Ethyl4-(4-N-(tert-butoxycarbonyl)-amino-3-fluorophenoxy)-2-aminopyridin-3-yl-carbamate(5a) (1.13 g, 2.8 mmol) was dissolved in TEA (8 mL), a few drops ofwater were added and the reaction mixture was stirred for 2 hours atroom temperature. The TEA was evaporated, the residue dissolved in water(20 mL), neutralized with saturated aqueous Na₂CO₃ and extracted withDCM (2×20 mL). The organic layer was dried and evaporated to afford thetitle compound.

Yield: 730 mg, 86%. ¹H-NMR δ: 1.17 (t, 3H, J=7.1, CH_(3.Et)), 4.05 (q,2H, J=7.0, CH_(2,Et)), 5.04 (s, 2H, NH_(2,Ph)), 5.71 (s, 2H, NH_(2,Py)),5.86 (d, 1H, J=5.7, H_(Py)), 6.64 (d, 1H, H_(arom)), 6.73-6.82 (m, 2H,H_(arom)), 7.68 (d, 1H, J=5.7, H_(Py)), 8.23 (br s, 1H, NH_(Py3)).LC-MS: m/z 307 ([M+H]⁺, 100).

Synthesis 6 Ethyl4-(4-amino-3-fluorophenoxy)-2-aminopyridin-3-yl-methyl-carbamate (7a)

Ethyl 4-(4-amino-3-fluorophenoxy)-2-aminopyridin-3-yl-carbamate (6a)(480 mg, 1.6 mmol) was dissolved in dry THF (8 mL) and cooled at 0° C.Sodium hydride (60% in mineral oil, 80 mg, 2.0 mmol) was added, and thereaction mixture was stirred for 40 minutes at 0° C. Methyl iodide (130μL, 1.8 mmol) was added at 0° C. The ice bath was removed, and themixture was stirred at room temperature for 18 hours. The solvent wasevaporated and the residue partitioned between DCM and distilled water.The organic layer was dried and evaporated, and the residue washed withdiethyl ether to afford the title compound as a brown solid.

Yield: 322 mg, 63%. ¹H-NMR δ: 1.09 (t, 3H, J=7.0, CH_(3.Et)), 3.00 (s,3H, CH₃N), 3.90-4.10 (m, 2H, CH_(2,Et)), 5.07 (s, 2H, NH_(2,Ph)), 5.87(d, 1H, Hp), 6.03 (s, 2H, NH_(2,Py)), 6.63 (t, 1H, H_(arom)) 6.77-6.81(m, 2H, H_(arom)), 7.75 (d, 1H, H_(Py)).

Synthesis 77-(4-Amino-3-fluorophenoxy)-1-N-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one(8a)

Ethyl 4-(4-amino-3-fluorophenoxy)-2-aminopyridin-3-yl-methyl-carbamate(7a) (320 mg, 1.0 mmol) was suspended in a solution of EtONa in EtOH (4mL), obtained from dissolving sodium (480 mg, 21 mmol) in ethanol (9mL). The suspension was heated under microwave irradiation for 1 hour(100° C., 100 W). The mixture was cooled at room temperature and thesolvent was evaporated. The residue was dissolved in water and wasacidified with AcOH to pH 4. The precipitate formed was recovered byfiltration, to afford the title compound.

Yield: 188 mg, 67%. ¹H-NMR δ: 3.46 (s, 3H, CH₃N), 5.11 (s, 2H, NH₂),6.35 (d, 1H, J=5.9, H_(Py)), 6.77-6.82 (m, 2H, H_(arom)), 6.99 (d, 1H,H_(arom)), 7.76 (d, 1H, J=6.0, Hp), 11.54 (s, 1H, NH_(Py)).

Synthesis 81-(3-Tert-butyl-1-phenyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(1-N-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenyl)urea(AA-01)

3-Tert-butyl-1-phenyl-1H-pyrazol-5-amine (550 mg, 1.8 mmol) wasdissolved in CH₂Cl₂ (25 mL) and an equal volume of saturated NaHCO₃ (aq)was added. The biphasic mixture was stirred and cooled to 0° C. with anice/water bath. After 10 minutes, 2 equiv. of a 1.9 M solution ofphosgene in toluene were added. The mixture was vigorously stirred for10 minutes, the organic layer was isolated, washed with H₂O, dried(MgSO₄) and concentrated to about 5 mL. This solution was added to asolution of the7-(4-amino-3-fluorophenoxy)-1-N-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one(8a) (200 mg, 1.4 mind) in THF. The solution was stirred for 15 hours atroom temperature, the solvents were evaporated and the solid residue waswashed with Et₂O and CH₂Cl₂ to afford the title compound as a whitesolid.

Yield: 200 mg, 53%. ¹H NMR, δ: 1.28 (s, 9H, tert-Bu), 3.42 (s, 3H, CH₃),6.39 (s, 1H, H_(Pyz,4)), 6.49 (d, 1H, J=5.9, H_(Py,5)), 6.99 (dd, 1H,J=1.6, 9.0, H_(arom)), 7.21 (dd, 1H, J=11.9, 2.7, H_(arom)), 7.40-7.45(m, 1H, H_(arom)), 7.50-7.58 (m, 4H, H_(arom)), 7.81 (d, 1H, J=5.9,H_(Py,6)), 8.10 (t, 1H, J=9.1, H_(arom)), 8.80 (s, 1H, NH_(urea)), 8.93(s, 1H, NH_(urea)), 11.63 (bs, 1H, NH_(Py2)). LC-MS: m/z 516 ([M]⁺,100). HRMS (El): m/z calcd for C₂₇H₂₇N₇O₃ ([M+H]⁺): 516.2154; found:516.2152.

Synthesis 91-(3-Tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(1-N-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenyl)urea(AA-02)

The title compound was prepared from3-tertbutyl-1-p-tolyl-1H-pyrazol-5-amine (458 mg, 2 mmol) and7-(4-amino-3-fluorophenoxy)-1-N-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one(8a) (110 mg, 0.4 mmol) by the same method as described for (AA-01), asan off-white solid.

Yield: 150 mg, 71%. ¹H NMR, δ: 1.27 (s, 9H, tert-Bu), 2.38 (s, 3H,Ph-CH₃), 3.42 (s, 3H, N-CH₃), 6.37 (s, 1H, H_(Pyz,4)), 6.49 (d, 1H,J=5.9, H_(Py,5)), 6.96 (dd, 1H, H_(arom)), 7.20 (d, 1H, H_(arom)), 7.34(d, 2H, J=8.3, H_(arom)), 7.39 (d, 2H, J=8.4, H_(arom)), 7.81 (d, 1H,J=5.9, H_(Py,6)), 8.11 (t, 1H, H_(arom)), 8.74 (s, 1H, NH_(urea)), 8.92(s, 1H, NH_(urea)), 11.61 (bs, 1H, NH_(Py2)). LC-MS: m/z 530 ([M+H]⁺,100).

Synthesis 101-(3-Tert-butyl-1-(4-chlorophenyl)-1H-pyrazol-5-yl)-3-(2-fluoro-4-(1-N-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenyl)urea(AA-03)

The title compound was prepared from3-tert-butyl-1-(4-chlorophenyl)-1H-pyrazol-5-amine (375 mg, 1.5 mmol)and7-(4-amino-3-fluorophenoxy)-1-N-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one(8a) (150 mg, 0.55 mmol) by the same method as described for (AA-01), asan off-white solid.

Yield: 190 mg, 63%. H ¹NMR, δ: 1.28 (s, 9H, tert-Bu), 3.42 (s, 3H, CH₃),6.39 (s, 1H, H_(Pyz,4)), 6.49 (d, 1H, J=5.9, H_(Py,5)), 6.96 (dd, 1H,9.0, H_(arom)),7.21 (d, 1H, H_(arom)), 7.57 (d, 2H, J=9.0, H_(arom)),7.60 (d, 2H, J=8.9, H_(arom)), 7.81 (d, 1H, J=5.9, H_(Py,6)), 8.08 (t,1H, H_(arom)), 8.80 (s, 1H, NH_(urea)), 8.89 (s, 1H, NH_(urea)), 11.63(bs, 1H, NH_(Py2)). LC-MS: m/z 549 ([M]⁺, 100).

Synthesis 11 3-Tert-butyl-1-(3-fluorophenyl)-1H-pyrazole-5-carboxylicacid

In a round-bottomed flask (dried in an oven) (3-fluorophenyl) boronicacid (224 mg, 1.6 mmol), ethyl-3-t-butyl-pyrazol-5-carboxylate (320 mg,1.6 mmol), copper acetate (355 mg, 1.9 mmol) and dry pyridine (158 μL,1.9 mmol) were suspended under vigorous stirring and an argon atmospherein 10 mL dry DMF. To the reaction mixture, 300 mg of 4 A molecular sievewas added and the suspension stirred for 20 hours at room temperature.The suspension was diluted with 20 mL AcOEt, washed with water (2×20mL), then with 20 mL conc. NaHCO₃ solution, and 20 mL brine, dried(MgSO₄) and evaporated under vacuum. A thick oil was obtained (520 mg)which was used in the next step without purification.

The oil was dissolved in 10 mL EtOH and 3 mL NaOH solution (2 M) wasadded under stirring and the reaction mixture was refluxed for 30minutes. After cooling to room temperature, the reaction mixture wasadjusted to pH 4 (with AcOH) and extracted with 20 mL AcOEt. The organiclayer was washed with water (2×20 mL), dried and evaporated undervacuum. A solid was obtained (457 mg). The solid was purified on Biotageusing cyclohexane:AcOEt 3:1 to afford the title compound as a whitesolid.

Yield: 166 mg (40%). ¹ H NMR (500 MHz, DMSO-d₆): δ 1.30 (s, 9H), 6.95(s, 1H, H_(pyr)), 8.59 (d, 1H, J=8.6 Hz), 7.25-7.32 (m, 1H, H_(arom)),7.35 (d, 1H, J=9.9 Hz, H_(arom)), 7.47-7.52 (m, 1H, H_(arom)), 13.22 (s,1H, H_(acid)). HRMS: (M+H)⁺ calcd for C₁₄H₁₅FN₂O₂, 262.1118, found:262.1117.

Synthesis 12 1-(3-Tert-butyl-1-(3-fluorophenyl)-1H-pyrazol-5-yl)-3-(2-fluoro-4-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenyl)urea(AA-04)

3-Tert-butyl-1-(3-fluorophenyl)-1H-pyrazole-5-carboxylic acid (393 mg,1.5 mmol) was dissolved in dry DMF (8 mL), and triethylamine (209 μL,1.5 mmol) was added. The solution was cooled at 0° C., anddiphenylphosphoryl azide (323 μL, 1.5 mmol) was added. The reactionmixture was stirred for 30 minutes at 0° C., followed by 1 hour at roomtemperature.7-(4-Amino-3-fluorophenoxy)-1-N-thyl-1H-imidazo[4,5-b]pyridin-2(3H)-one(8a) (140 mg, 0.5 mmol) was added, and the reaction mixture heated at110° C. for 1 hour. The solution was cooled, diluted with AcOEt (100 mL)and extracted with water and brine. The organic layer was dried andevaporated, and the residue taken up in DCM. The remaining solid wasrecovered by filtration to afford the title compound.

Yield: 25 mg, 9%. ¹H NMR, δ: 1.28 (s, 9H, tert-Bu), 3.41 (s, 3H, CH₃),6.40 (s, 1H, H_(Pyz,4)), 6.49 (d, 1H, J=6.1, H_(Py,5)), 6.96-7.02 (m,1H, H_(arom)), 7.19-7.26 (m, 2H, H_(arom)), 7.36-7.44 (m, 2H, H_(arom)),7.58 (t, 1H, H_(arom)), 7.81 (d, 1H, J=5.9, H_(Py,6)), 8.06 (t, 1H,J=9.1, H_(arom)), 8.83 (s, 1H, NH_(urea)), 8.92 (s, 1H, NH_(urea)),11.64 (bs, 1H, NH_(Py2)). LC-MS: m/z 533 ([M]⁺, 100).

Synthesis 13Tert-butyl-4-(2-amino-3-nitropyridin-4-yl-oxy)naphthalen-1-yl-carbamate(3b)

The title compound was prepared from tert-butyl4-hydroxynaphthalen-1-ylcarbamate (Regan, J. et al, J. Med. Chem., 2002,Vol. 45, No. 14, p. 2994) (3.9 g, 15 mmol) by the same method asdescribed for compound (3a).

Yield: 5.4 g, 90%, upon recrystallization from dichloromethane. ¹H-NMR(DMSO-d₆), δ (ppm), J (Hz): 1.52 (s, 9H, C(CH₃)₃), 5.80 (d, 1H, J=5.7,PyrH), 7.26 (s, 2H, NH₂), 7.38 (d, 1H, J=8.3, ArH,Naph), 7.58-7.69 (m,3H, ArH, Naph), 7.86-7.89 (m, 1H, ArH(Naph), 7.93 (d, 1H, J=5.5, PyrH),8.14-8.17 (m, 1H, ArH, Naph), 9.36 (s, 1H, NHBoc).

Synthesis 14Tert-butyl-4-(2,3-diaminopyridin-4-yl-oxy)naphthalen-1-yl-carbamate (4b)

The title compound was prepared fromtert-butyl-4-(2-amino-3-nitropyridin-4-yl-oxy)naphthalen-1-yl-carbamate(3b) (0.50 g, 1.26 mmol) by the same method as described for compound(4a) as a brown solid.

Yield: 0.38 g (82%). ¹H-NMR (DMSO-d₆), δ (ppm), J (Hz): 1.55 (s, 9H,C(CH₃)₃), 4.63 (s, 2H, NH₂), 5.66 (s, 2H, NH₂), 5.92 (d, 1H, J=5.6,PyrH), 7.05 (d, 1H, J=8.3, ArH,Naph), 7.24 (d, 1H, J=5.5, PyrH), 7.54(d, 1H, J=8.3, ArH,Naph), 7.60-7.65 (m, 2H, ArH, Naph), 8.07-8.12 (m,2H, ArH, Naph), 9.22 (s, 1H, NHBoc).

Synthesis 154-(4-N-Boc-aminonaphthalen-1-yl-oxy)-3-N-aminocarbamoylethyl-2-amino-pyridine(5b)

4-(4-N-Boc-aminonaphthalen-1-yl-oxy)-2,3-diaminopyridine (4b) (500 mg,1.4 mmol) and the pyridine (222 μL, 2.7 mmol) were dissolved in dry THF(8 mL) under vigorous stirring at 0° C. To this solution theethylchloroformate (136 mL, 1.5 mmol) was added at once. The reactionmixture was allowed to reach room temperature and was stirred for anadditional 10 hours. The solvent was evaporated under vacuum and theresidue partitioned between EtOAc and Na₂CO₃ solution. The organic layerwas washed (20 mL brine), dried (MgSO₄) and evaporated to provide asolid residue. After purification by LC (Isolute column, Flash Si II, 50g/170 mL; eluent: EtOAc), the desired compound was obtained.

Yield: 475 mg, 75%. ¹H-NMR δ: 1.14-1.21 (m, 3H, CH₃), 1.50 (s, 9H, tBu),4.04-4.10 (m, 2H, CH₂), 5.66 (d, 1H, J=5.7, H_(Py)), 5.84 (s, 2H, NH₂),7.15 (d, 1H, J=8.1, H_(arom)), 7.49-7.60 (m, 3H, H_(arom)), 7.62 (d, 1H,J=5.7, H_(Py)), 7.98-8.04 (m, 1H, H_(arom)), 8.07 (d, 1H, J=8.5,H_(arom)), 8.40 (s, 1H, NH_(carb)) 9.22 (s, 1H, NH_(Boc)). LC-MS: m/z440 [(M+H)⁺, 100]. HRMS, (El): m/z calcd for C₂₃H₂₇N₄O₅[(M+H)⁺]:439.1981; found 439.1979.

Synthesis 164-(4-Aminonaphthalen-1-yl-oxy)-3-N-aminocarbamoylethyl-2-amino-pyridine(6b)

4-(4-N-Boc-aminonaphthalen-1-yl-oxy)-3-N-aminocarbamoylethyl-2-aminopyridine(5b) (475 mg, 1.05 mmol) was dissolved in dry TFA (10 mL) under vigorousstirring at 0° C. The solution was allowed to reach room temperature andwas stirred for an additional 2 hours. The TFA was evaporated undervacuum and the oily residue partitioned between EtOAc and Na₂CO₃solution. The organic layer was washed (20 mL brine), dried (MgSO₄) andevaporated to provide a solid residue.

Yield: 346 mg, 97%. ¹H-NMR δ: 1.19-1.26 (m, 3H, CH₃), 4.07-4.13 (m, 2H,CH₂), 5.57 (d, 1H, J=5.7, H_(Py)), 5.77 (s, 4H, 2×NH₂), 6.66 (d, 1H,J=8.1, H_(arom)), 6.97 (d, 1H, J=8:1, H_(arom)), 7.37-7.45 (m, 2H,H_(arom)), 7.55 (d, 1H, J=5.7, H_(Py)), 7.76-7.86 (bs, 1H, H_(arom)),8.09.-8.12 (m, 1H, H_(arom)), 8.36 (s, 1H, NH_(carb)). LC-MS: m/z 339[(M+H)⁺, 100]. HRMS (El): m/z calcd for C₁₃H₁₉N₄O₃[(M+H)⁺, 100]:339.1457; found 339.1459.

Synthesis 174-(4-Aminonaphthalen-1-yl-oxy)-3-N-methyl-N-aminocarbamoylethyl-2-amino-pyridine

4-(4-Aminonaphthalen-1-yl-oxy)-3-N-aminocarbamoylethyl-2-amino-pyridine(6b) (350 mg, 1.04 mmol) was dissolved in dry THF (8 mL) under vigorousstirring at 0° C. and argon. To this solution NaH (60% dispersed inmineral oil) (45 mg, 1.14 mmol) was added. After 40 minutes, Mel (66 mL,0.91 mmol) was added at 0° C. The reaction mixture was allowed to reachroom temperature and was stirred for further 10 hours. The solvent wasevaporated under vacuum and the residue retaken in 20 mL of EtOAc. Thesolution was washed with brine (2×20 mL), dried and evaporated todryness. The residue was triturated with Et₂O and filtered to give thetitle compound as a solid.

Yield: 238 mg, 65%. ¹H-NMR δ: 1.12-1.29 (m, 3H, CH₃), 3.14 (s, 3H, CH₃),4.05-4.16 (m, 2H, CH₂), 5.53 (d, 1H, J=5.8, H_(Py)), 5.75 (s, 2H, NH₂),6.01 (s, 2H, NH₂), 6.66 (d, 1H, J=8.1, H_(arom)), 6.96 (d, 1H, J=8.1,H_(arom)), 7.37-7.43 (m, 2H, H_(arom)), 7.57 (d, 1H, J=5.8, H_(Py)),7.59-7.64 (m, 1H, H_(arom)), 8.11-8.15 (m, 1H, H_(arom)). LC-MS: m/z 353([M+H]⁺, 100). HRMS (El): m/z calcd for C₁₉H₂₁N₄O₃([M+H]⁺): 353.1614;found 353.1610.

Synthesis 187-(4-Aminonaphthalen-1-yl-oxy)-1-N-methyl-1-H-imidazo[4,5-1]pyridine-2(3H)-one(8b)

230 mg (0.65 mmol)4-(4-aminonaphthalen-1-yl-oxy)-3-N-methyl-N-aminocarbamoylethyl-2-aminopyridine(7b) were suspended in 5.0 mL solution 1.0 M of EtONa in EtOH. Thesuspension was submitted to microwave (150 W, 100° C.) for 45 minutes.After cooling, the reaction mixture was evaporated to dryness, retakenin 20 mL H₂O, the pH adjusted to 4.5 (AcOH), precipitating the titlecompound.

Yield: 167 mg, 84%. ¹H-NMR δ: 3.61 (s, 3H, CH₃), 5.79 (s, 2H, NH₂), 6.10(d, 1H, J=5.9, F_(Py)), 6.68 (d, 1H, J=8.1, H_(arom)), 7.10 (d, 1H,J=8.1, H_(arom)), 7.43-7.48 (m, 2H, H_(arom)), 7.64 (d, 1H, J-5.8,H_(Py)), 7.74-7.81 (m, 1H, 8.12-8.17 (m, 1H, H_(arom)), 11.57(s, 1H,NH_(Py)). LC-MS: m/z 307 ([M+H]⁺, 100). HRMS (El): m/z calcd forC₁₇H₂₅N₄O₂ ([M+H]⁺): 307.1195; found 307.1188.

Synthesis 19 1-(3-Tert-butyl-1 -phenyl-1H-pyrazol-5-yl)-3-(4-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl-oxy)naphthalen-1-yl)urea(BB-01)

The title compound was prepared from3-tert-butyl-1-phenyl-1H-pyrazol-5-amine (0.18 mmol) and7-(4-aminonaphthalen-1-yl-oxy)-1-N-methyl-1H-imidazo[4,5-b]pyridine-2(3H)-one(8b) (50 mg, 0.16 mmol) by the same method as described for (AA-01), asan off-white solid.

Yield: 87 mg, 98%. ¹H NMR, δ 1.29 (s, tert-Bu), 3.53 (s, 3H, CH₃), 6.29(d, 1H, J=5.9, H_(Py,5)), 6.40 (s, 1H, H_(pyr)), 7.24 (d, 1H, J=8.3,H_(arom)), 7.43 (t, 1H, J=7.0 Hz), 7.54-7.63 (m, 5H), 7.66 (t, 1H,J=8.2, H_(arom)), 7.74 (d, 1H, J=5.9, H_(Py,6)), 7.86 (d, 2H, Jμ8.3,H_(arom)), 8.05-8.10 (m, 1H), 8.77 (s, 1H, 9.07 (s, 1H, NH_(urea)),11.64(S, 1H, NH_(Py2)). LC-MS: R_(f)=8.25 min; m/z 548.2 (M⁺, 100). HRMS(El): m/z calcd for C₃₁ H₃₀N₇O₃ ([M—H]⁺): 548.2410; found: 548.2404.

Synthesis 201-(1-N-p-tolyl-3-tert-butyl-pyrazol-5-yl)-3-(4-(2-oxo-2,3-dihydro-1-N-methyl-1H-imidazo[4,5-b]pyridin-7-yl-oxy)-naphthalen-1-yl)urea(BB-02)

The title compound was prepared from3-tertbutyl-1-p-tolyl-1H-pyrazol-5-amine (35 mg, 0.15 mmol) and7-(4-aminonaphthalen-1-yl-oxy)-1-N-methyl-1H-imidazo[4,5-b]-pyridin-2(3H)-one(8b) (40 mg, 0.13 mmol) by the same method as described for (AA-01), asan off-white solid.

Yield: 52 mg, 71%. ¹H NMR, δ: 1.28 (s, 9H, tert-Bu), 2.40 (s, 3H, CH₃),3.53 (s, 3H, CH₃), 6.29 (d, 1H, J=5.9, H_(Py.5)), 6.39 (s, 1H, H_(pyr)),7.24 (d, 1H, J=8.4, H_(arom)), 7.36 (d, 2H, J=8.2, H_(arom)), 7.45 (d,2H, J=8.2, H_(arom)), 7.60 (t, 1H, J=7.5, H_(arom)), 7.67 (t, 1H, J=7.6,H_(arom)), 7.74 (d, 1H, J=5.9, H_(Py,6)), 7.87 (d, 1H, J=8.3, H_(arom)),8.07 (d, 2H, J=8.3, H_(arom)), 8.71 (s, 1H, NH_(urea)) 9.06 (s, 1H, NH),11.65 (s, 1H, NH_(Py3)). LC-MS: R_(t)=5.23 min; m/z 562.2 ([M+H]⁺, 100).HRMS (El): m/z calcd for C₃₂H₃₂N₇O₃ ([M+H]⁺): 562.2561; found: 562.2566.

(Reference) Synthesis 21 Tert-butyl2-fluoro-4-(2-(methylamino)-3-nitropyridin-4-yloxy)phenylcarbamate

Tert-butyl 2-fluoro-4-hydroxyphenylcarbamate (3.25 g, 14.4 mmol) wasdissolved in DMSO (25 mL) and the solution was stirred under an argonatmosphere for 20 minutes. Sodium hydride (60% in mineral oil, 580 mg,14.4 mmol) was added portionwise and the dark solution was stirred atroom temperature for 1 hour. 4-chloro-N-methyl-3-nitropyridin-2-amine(2.7 g, 14.4 mmol) dissolved in DMSO (5 mL) was added at once and thered solution was stirred at 50′C for 2 hours. The solution was cooled,poured onto crushed ice (200 g) and extracted with ethyl acetate (3×100mL). The organic phases were washed with brine, dried and evaporated togive the title compound as a yellow solid.

Yield: 5.3 g, 90%. ¹H NMR (500 MHz, DMSO-d₆) δ: 1.47 (s, 9H, tert-Bu)2.93 (d, J=4.5 Hz, CH₃N), 6.08 (d, J=57 Hz, 1H, H_(py)), 7.00 (m, 1H,H_(arom)), 7.22 (m, 1H, H_(arom)), 7.55 (q, J=4.5 Hz, 1H, NHCH₃), 7.66(m, 1H, H_(arom)), 8.14 (d, J=5.7 Hz, 1H, 1H, H_(py)), 9.04 (bs, 1H,NH). LC-MS: m/z 378.3 ([M+H]⁺, 100).

(Reference) Synthesis 22 Tert-butyl4-(3-amino-2-(methylamino)pyridin-4-yloxy)-2-fluorophenylcarbamate

Tert-butyl2-fluoro-4-(2-(methylamino)-3-nitropyridin-4-yloxy)phenylcarbamate (5.3mg, 14 mmol) was dissolved in absolute ethanol (600 mL) and hydrogenatedon a 10% Pd/C cartridge through an H-Cube apparatus, to give the titlecompound as a yellow solid.

Yield: 4.88 g (quantitative yield). ¹H NMR (500 MHz, DMSO-d₆) δ: 1.44(s, 9H, tert-Bu), 2.85(d, 3H), J=4.5 Hz, CH₃N), 4.51 (bs, 2H, NH₂), 5.94(m, 1H, CH₃NH), 6.10 (d, 1H, J=5.6 Hz, H_(py)), 6.72 (m, 1H, H_(arom)),6.85 (m, 1H, H_(arom)), 7.37 (d, 1H, J=5.6 Hz, H_(p)), 7.44 (m, 1H,H_(arom)), 8.87 (bs, 1H, NH). LC-MS: m/z 348.4 ([M+H]⁺, 100).

Reference Synthesis 23 Tert-butyl2-fluoro-4-(3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenylcarbamate

To an ice-cooled solution of tert-butyl4-(3-amino-2-(methylamino)pyridin-4-yloxy)-2-fluorophenylcarbamate (4.9g, 14 mmol) in THF (150 mL) and pyridine (10 mL) under an argonatmosphere, a solution of triphosgene (4.45 g, 15 mmol) in THF (75 mL)was added over 2 hours via a dropping funnel. The solution was stirredfor 2 hours at 0° C., followed by 4 hours at room temperature, and thenrefluxed overnight. After cooling, the solution was filtered, evaporatedand chromatographed on a Biotage apparatus (25+M column, eluentDCM/EtOAc 1/1) to give the title compound as a white solid.

Yield: 2.05 g, 40%. ¹H NMR (500 MHz, DMSO-d₆) δ: 1.47 (s, 9H, tert-Bu),3.32 (s, 3H, CH₃N), 6.53 (d, 1H, J=5.9 Hz, H_(py)), 6.94 (m, 1H,H_(arom)), 7.15 (m, 1H, H_(arom)), 7.60 (m, 1H, H_(arom)), 7.89 (d, 1H,J=5.9 Hz, H_(py)), 8.97 (s, 1H, NH), 11.46 (s, 1H, NH). LC-MS: m/z 375.1([M+H]⁺, 100).

(Reference) Synthesis 247-(4-Amino-3-fluorophenoxy)-3-methyl-1H-imidazo[4,5-b ]pyridin-2(3H)-one

A solution of tert-butyl2-fluoro-4-(3-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)phenylcarbamate(2.18 g, 5.8 mmol) in 1 M TBAF in THF (41 mL) was refluxed overnightunder an argon atmosphere. The solution was cooled and evaporated andwater (20 mL) was added, upon which a precipitate formed. Theprecipitate was filtered, washed with water, and dried to give the titlecompound as an off-white solid.

Yield: 1.37 g, 86%. ¹H NMR (500 MHz, DMSO-d₆): δ 3.29 (s, 3H, CH₃N),5.17 (s, 2H, NH₂), 6.35 (d, 1H, J=5.94 Hz, H_(py)). 6.74-6.83 (m, 2H,H_(arom)), 6.99 (m, 1H, H_(arom)), 7.81 (d, 1H, J=5.94 Hz, H_(py)),11.44 (s, 1H, NH). LC-MS: m/z 275.1 ([M+H]⁺, 100).

(Reference) Synthesis 257-(4-amino-3-fluorophenoxy)-1,3-dimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-one

To a solution of7-(4-amino-3-fluorophenoxy)-3-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one(100 mg, 0.365 mmol) in THF (4 mL) at 0° C. under argon, NaH (16.77 mg,0.419 mmol) was added in one portion. The resulting solution was stirredfor 20 minutes and iodomethane (0.025 mL, 0.401 mmol) was added. After 1hour, water was added, and the solution evaporated and extracted withDCM (3×20 mL) to give the title compound.

Yield: 80 mg, 0.278 mmol, 76%. ¹H NMR (CDCl₃), δ: 3.51 (s, 3H, Me), 3.66(s, 3H, Me), 6.40 (d, 1H, J=6.0, H_(arom,Py)) 6.74 (ddd, 1H, J=8.6, 2.5,1.0, H_(arom,FPh)), 6.83 (m, 2H, H_(arom,FPh)) 7.87 (d, 1H, J=6.0,H_(arom,Py)). LC-MS: Rf=2.60 min; m/z 289.1 ([M+H]+, 90).

(Reference) Synthesis 261-(1-N-p-tolyl-3-tert-butyl-pyrazol-5-yl)-3-(4-(2-oxo-2,3-dihydro-1-N-methyl-1H-imidazo[4,5-b]pyridin-7-yl-oxy)phenyl)urea(XX-02)

The title compound was prepared using3-tert-butyl-1-p-tolyl-1H-pyrazol-5-amine (43.7 mg, 0.19 mmol) and7-(4-aminophenyloxy)-1-N-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one(Niculescu-Duvaz, D. et al, J. Med. Chem., 2009, Vol. 52, No. 8, p.2255) (40 mg, 0.16 mmol) by the same method as described for (AA-01), asa white solid.

Yield: 62 mg, 76%. ¹H NMR, δ: 1.27 (s, 9H, tert-Bu), 2.37 (s, 3H, CH₃),3.44 (s, 3H, CH₃), 6.35 (d, 1H, J=5.9, H_(Py.5)), 6.39 (s, 1H, H_(pyr)),7.11 (d, 2H, J=9.0, H_(arom)), 7.33 (d, 2H, J=8.3, H_(arom)), 7.39 (d,2H, J=8.3, H_(arom)), 7.47 (d, 2H, J=9.0, H_(arom)), 7.78 (d, 1H, J=5.9,H_(Py,6)), 8.32 (s, 1H, NH_(urea)), 9.08 (s, 1H, NH_(urea)), 11.59 (s,1H, NH_(Py3)). LC-MS: R_(f)=5.14 min; m/z 511 ([M+H]⁺, 100). HRMS (El):m/z calcd for C₂₃H₃₀N₇O₃ ([M+H]⁺): 512.2405; found: 512.2405.

(Reference) Synthesis 271-(3-Tert-butyl-1-phenyl-1H-pyrazol-5-yl)-3-(4-(1,3-dimethyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)-2-fluorophenyl)urea(XX-03)

The title compound was prepared from3-tert-butyl-1-phenyl-1H-pyrazol-5-amine (130 mg, 0.60 mmol) and7-(4-amino-3-fluorophenoxy)-1,3-dimethyl-1H-imidazo[4,5-b]pyridin-2(3H)-one(100 mg, 0.35 mmol) by the same method as described for (AA-01), as ayellow powder.

Yield: 15 mg, 8%. ¹H NMR (CDCl₃), δ: 1.39 (s, 9H, tert-Bu), 3.48 (s, 3H,Me), 3.59 (s, 3H, Me), 6.47 (d, 1H, J=5.9, H_(arom,Py)), 6.49 (s, 1H,H_(Pyz,4)), 6.85 (dd, 1H, J=11.1, 2.5, H_(arom,FPh)), 6.89 (d, 1H,J=9.0, H_(arom,FPh)), 7.31 (t, 1H, J=7.7, H_(arom,Ph)), 7.42 (t, 2H,J=7.7, H_(arom,Ph)), 7.50 (d, 2H, J=7.9, H_(arom,Ph)) 7.92 (d, 1H,J=5.9, H_(arom,Py)), 8.15 (t, 1H, J=8.9, H_(arom,FPh)) LC-MS: Rf=2.78min; m/z 530 ([M+H]+, 90). HRMS (El): m/z calcd for C₂₈H₂₉FN₇O₃([M+H]+): 530.2310; found: 530.2326.

Reference Synthesis 281-(5-Tert-butyl-2-phenyl-2H-pyrazol-3-yl)-3-[4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)-phenyl]-urea(XX-01)

The title compound was obtained using known methods, as shown, forexample, in Synthesis 61 in Niculescu-Duvaz et al., 2006.

(Reference) Synthesis 29 1-[5-tert-Butyl-2-(4-fluoro-phenyl)-2H-pyrazol-3-yl]-3-[4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)-phenyl]-urea(XX-04)

The title compound was obtained using known methods, as shown, forexample, in Synthesis 79 in Niculescu-Duvaz et al., 2006.

Biological Methods Biological Methods—Assay A—DELFIA Kinase Assay

Compounds were assessed by a kinase assay performed according to thefollowing protocol.

The following reagents were prepared:

DELFIA Kinase Buffer (DKB):

TABLE 2 Volume Volume per Stock per mL 10 ml plate Reagent Concentration(μL) (μL) 20 mM MOPS pH 7.2 0.2M 100 1000 0.5M EGTA pH 8.0 0.5M 10 10010 mM MgCl₂   1M 10 100 0.1% β-mercaptoethanol — 1 10 25 mMβ-glycerophosphate 0.5M 50 500 Water 100% 829 8290

MOPS=3-[N-Morpholino] propanesulfonic acid (Sigma M3183).

EGTA=Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid(Sigma E3889).

DKB1 (DKB with B-RAF and MEK protein):

Combine 4950 μL of DKB and 50 μL of 2.5 mg/mL GST-MEK stock (t) give 1mg of MEK per 40 μL). Then add 22.5 μL of B-RAF to give ˜0.2 μL of B-RAFper 40 μL.

DKB2 (DKB with MEK protein):

Combine 4950 μL of DKB and 50 μL of 2.5 mg/mL GST-MEK stock (to give 1mg of MEK per 40 μL). Use 500 μL of this for the blow out (BO) and theempty vector (EV) control.

ATP:

100 mM stock, dilute to 500 μM to give 100 μM final concentration inassay.

Inhibitors (Test Compounds):

100 mM stock, dilute to 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001,0.0003, and 0.0001 mM in DMSO in drug plate, resulting in concentrationof 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 μM in theassay.

Primary antibody:

Phospho-MEK1/2 CST #9121S diluted 1:1000 in DELFIA assay buffer (AB).Pre-incubate antibody in the AB for 30 minutes at room temperature priorto use.

Secondary antibody:

Anti-rabbit-Eur labelled secondary Perkin Elmer #AD0105 diluted 1:1000in DELFIA assay buffer (AB). Pre-incubate antibody in the AB for 30minutes at room temperature prior to use. (Primary and secondaryantibodies were incubated together.)

Tween:

0.1% Tween 20 in water.

Assay Buffer:

DELFIA assay buffer Perkin Elmer #4002-0010.

Enhancement Solution:

DELFIA enhancement solution Perkin Elmer #4001-0010.

Assay Plates:

96 well glutathione-coated black plate Perbio #15340.

Procedure:

1. Preblock wells with 5% milk in TBS for 1 hour.

2. Wash wells 3× with 200 μL TBS.

3. Plate out 40 μL of DKB1 for all inhibitors (test compounds), DMSOcontrol, and optionally other control compounds.

4. Plate out 40 μL of DKB2 for BO and EV wells.

5. Add inhibitors (test compounds) at 0.5 μL per well according todesired plate layout.

6. Add 0.5 μL DMSO to vehicle control wells.

7. Add 2 μL of B-RAF to BO and EV wells.

8. Pre-incubate with inhibitors (test compounds) for 10 minutes at roomtemperature with shaking.

9. Add 10 μL of 500 μM ATP stock, in DKB, to give 100 μM assayconcentration.

10. Seal plates with TopSeal and incubate at room temperature withshaking for 45 minutes.

11. Wash plates 3× with 200 μL 0.1% Tween20/Water to terminate reaction.

12. Add 50 μL per well of antibody mix and incubate for 1 hour at roomtemperature with shaking.

13. Wash plates 3× with 200 μL 0.1% Tween20/Water.

14. Add 100 μL DELFIA enhancement solution per well, cover in foil, andincubate at room temperature for 30 minutes with shaking.

15. Read on Victor using Europium protocol.

Values for the blank (Empty Vector) are subtracted from all values. TheDMSO controls are set as 100% activity and assay points (the respons()are calculated as a percentage of the DMSO control. Data are plottedusing Graphpad Prism software and a non-linear regression line iscalculated using a variable slope sigmoidal dose-response equation:

Y=Bottom+[Top−Bottom]/[1+10̂(LogEC50−X)*HillSlope)]

where X is the logarithm of concentration and Y is the response. TheIC50 generated by this procedure is the concentration of the drug thatproduces a percentage control fluorescence value midway between thesaturation, and zero-effect plateaus. Three independent assays areusually performed and the mean IC50 is reported.

Biological Methods—Assay B—Cell Based Phosho-ERK Assay

Compounds were assessed using a cell-based assay which was performedaccording to the following protocol.

Day 0:

Plate out 16,000 mutant BRAF WM266.4 cells/well in 99 μL medium in a96-well plate.

Day 1:

1. Add 1 μL inhibitor (test compound) to the cells (total 1 μLsolution).

2. Incubate the cells with test compound for 6 hours at 37° C.

3. Aspirate off the solution from all of the wells.

4. Fixate the cells with 100 μL 4% formaldehyde/0.25% Triton X-100 PBSper well.

5. Incubate the plate for 1 hour at 4° C.

6. Aspirate off the fixing solution and add 300 μL TBS per well.

7. Leave the plate overnight at 4° C.

Day 2:

1. Wash the plate 2× with 200 μL PBS per well.

2. Block with 100 μL 5% dried milk in TBS.

3. Incubate the plate for 20 minutes at 37° C.

4. Wash the plate 2× with 0.1% tween/H₂O.

5. Add 50 μL of 3 μg/mL primary antibody pERK (Sigma M8159), diluted in5% milk powder/TBS, to each well.

6. Incubate the plate for 2 hours at 37° C.

7. Wash the plate 3× with 0.1% tween/H₂O.

8. Add 50 μL of 0.45 μg/mL secondary Europium-labelled anti-mouseantibody (Perkin Elmer) to each well.

9. Incubate the plate for 1 hour at 37° C.

10. Wash the plate 3× with 0.1% tween/H₂O.

11. Add 100 μL enhancement solution (Perkin Elmer) to each well.

12. Leave the plate for approximately 10 minutes at room temperaturebefore gently shaking the plate.

13. Read Europium Time Resolved Fluorescence in Victor2.

14. Wash the plate 2× with 0.1% tween/H₂O.

15. Measure the protein concentration with BCA (Sigma) by adding 200 μLof solution per well.

16. Incubate the plate for 30 minutes at 37° C.

17. Read absorbance levels at 570 nm in a plate reader.

Note that Europium counts are normalised for protein levels by dividingcounts by absorbance.

Values for the blank (no cells) are subtracted from all values. The DMSOcontrols are set as 100% activity and assay points (the response) arecalculated as a percentage of the DMSO control. Data are plotted usingGraphpad Prism software and a non-linear regression line is calculatedusing a variable slope sigmoidal dose-response equation:

Y=Bottom+[Top−Bottom]/[1+10̂(LogEC50−X)*HillSlope)]

where X is the logarithm of concentration and Y is the response). TheIC50 generated by this procedure is the concentration of the drug thatproduces a percentage control fluorescence value midway between thesaturation, and zero-effect plateaus. Three independent assays areusually performed and the mean IC50 is reported.

Biological Methods—Assay C—SRB Cell Proliferation Assay (SRB GI₅₀)

Cell lines (e.g., WM266.4 and A375M melanoma cell lines; SW620colorectal carcinoma cell line) are routinely cultured in DMEM orRPMI1640 supplemented with 10% foetal bovine serum, at 37° C., in 10%CO₂ water saturated atmosphere. Cultures are maintained in exponentialgrowth phase by sub-culturing before having become confluent (3-5 dayintervals). Single cell suspensions are prepared by harvesting an 80 cm²tissue culture flask with 5 mL commercial trypsin EDTA. Alter 5 minutes,the detached cells are mixed with 5 fully complemented culture mediumand centrifugally pelleted (1000 rpm for 7 minutes). After aspiratingthe supernatant, the cell pellet is re-suspended in 10 mL fresh mediumand the cells fully disaggregated by drawing the whole volume up/down 5times through a 19-gauge needle. The concentration of the cells isdetermined using a haemocytometer ( 1/10 dilution). A suitable volume togive at least a 2-fold excess for the number of tests being conducted,typically 100-200 mL, is prepared by diluting the cell suspension to10,000-40,000/mL, and 100 μL/well dispensed into 96 well plates using aprogrammable 8-channel peristaltic pump, giving 1000-4000 cells/well,leaving column 12 blank. The plates are returned to the incubator for 24hours to allow the cells to re-attach.

The compounds being tested are prepared at 10 mM in DMSO. Aliquots (24μL) are diluted into 1.2 mL culture medium giving 200 μM, and 10 serialdilutions of 3× performed by transferring 80 μL to 160 μL. Aliquots (100μL) of each dilution are added to the wells, using an 8-channelpipettor, thus performing a final further 2× dilution, and giving dosesranging from 100 μM to 0.005 μM. Column 11 receives plain culture mediumonly. Each compound is tested in quadruplicate, each replicate being theaverage of four wells.

After a further 5 days growth, the plates are emptied, and the cells arefixed in 10% trichloroacteic acid for 30 minutes at 4° C. After thoroughrinsing in running tap water, the plates are dried, and stained byadding 50 μL of a solution of 0.1% sulphorhodamine-B in 1% acetic acid,for 10 minutes at room temperature. The stain is poured out and theplates thoroughly rinsed under a stream of 1% acetic acid, thus removingunbound stain, and dried. The bound stain is taken into solution byaddition of 100 μL Tris buffer pH 8, followed by 10 minutes on aplate-shaker (approximately 500 rpm). The absorbance at 540 nm in eachwell (being proportional to the number of cells present) is determinedusing a plate reader.

After averaging the blank values in column 12 this was subtracted fromall values, and results expressed as a percentage of the untreated value(column 11). The 10 values so derived (in quadruplicate) are plottedagainst the logarithm of the drug concentration, and analysed bynon-linear regression to a four parameter logistic equation, settingconstraints if suggested by inspection. The GI₅₀ generated by thisprocedure is the concentration of the drug that produces a percentagecontrol A₅₄₀ midway between the saturation, and zero-effect plateaus.

Biological Methods—Xenograft Studies

SW620 human colorectal carcinoma cells (mutant RAS, 7×10⁷) or A375Mhuman melanoma celss (mutant BRAF, 10⁷) were inoculated sub-cutaneuouslyin suspension (0.2 mL) into the right flank of female Crl:CD1-Foxn1nuathymic mice. Groups were assigned to treatment following stratifiedallocation of tumour volumes. Treatment with test compound began betweendays 11-14 post-cell administration. For gavage, a suspension(DMSO:water, 1:19, v/v at 10 mL/kg) was administered. Control animalsreceived a similar dosage of vehicle (DMSO : water, 1:19, v/v).Treatment with test compound was continued once daily for 24 doses.

Biological Data—Assay Data

Data for several compounds of the present invention, as well as severalcomparison compounds are summarised in the following table. LowerIC₅₀/GI₅₀ values indicate higher potency.

TABLE 3 Assay A Assay B Assay C Assay C Assay C BRAF pERK SRB SRB SRB —WM266.4 WM266.4 A375M SW620 Code IC₅₀ (μM) IC₅₀ (μM) GI₅₀ (μM) GI₅₀ (μM)GI₅₀ (μM) AA-01 0.27 0.26 0.052 0.138 0.167 AA-02 0.197 0.020 0.057AA-03 0.302 0.060 0.121 BB-01 0.25 0.019 0.008 0.052 1.6 BB-02 2.280.026 0.016 0.101 1.685 XX-01 0.094 0.62 0.39 1.13 0.84 XX-02 0.20 0.090.066 0.25 0.45 XX-03 1.09 0.12 0.033 0.247 6.83 XX-04 0.24 0.20 0.0200.307 2.72

In the in vitro BRAF enzyme assay (Assay A), the compounds of thepresent invention (˜0.2-2.3 μM) and the comparison compounds (˜0.1-1.1μM) had similar BRAF IC₅₀ values.

In the in vitro pERK cell-based assay (Assay B), the compounds of thepresent invention (˜0.02-0.26 μM) and the comparison compounds (˜0.1-0.6μM) had similar pERK IC₅₀ values.

In the in vitro SRB cell-based Assay (Assay C), for the mutant BRAF celllines WM266.4 and A375M, the compounds of the present invention(WM266.4: ˜0.01-0.12 μM; A375M: ˜0.05-0.14 μM) and the comparisoncompounds (WM266.4: 0.02-0.4 μM; A375M: ˜0.25-1.1 μM) had similar G150values. Notably, BR-01 was the most potent (with the lowest GI₅₀:WM266.4, 0.008 μM; A375M, 0.052 μM), and XX-01 was the least potent(with the highest GI₅₀: WM266.4, 0.39 μM; A375M, 1.13 μM).

In the in vitro SRB cell-based Assay (Assay C), for the mutant RAS cellline SW620, the compounds of the present invention (SW620: ˜0.17-1.6 μM)and the comparison compounds (SW620: ˜0.45-7 μM) had similar GI50values.

However, note that, based on the in vitro mutant RAS data, it would beexpected that XX-01 and XX-02 would have better therapeutic efficacythan BB-02, in the in vivo mutant RAS SW620 xenograft study.

Similarly, based on the in vitro mutant RAS data, it would be expectedthat XX-02 would have a similar therapeutic efficacy as AA-01, in the invivo mutant RAS SW620 xenograft study.

Based on the in vitro mutant RAS data, it would not be expected that theclaimed compounds (especially AA-01 and BB-02) are substantially moreeffective than the comparison compounds (XX-01, XX-02, XX-03, XX-04), inthe in vivo mutant RAS SW620 xenograft study.

Biological Data—BRAF/RAS/Wild Type Selectivity Data

Data for several compounds of the present invention, as well as severalcomparison compounds are illustrated in FIGS. 4 to 10, as discussedbelow. The data illustrate the selectivity, or lack of selectivity, formutant BRAF or mutant RAS cell lines.

FIG. 4 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for compound AA-01.The range of cell lines are (a) a panel of mutant BRAF (mutBRAF) celllines: WM266.4, A375M, UACC62; (b) a panel of mutant RAS (mutRAS) celllines: SW620, HCT116, and WM1361; and (c) a panel of wild type BRAF andRAS (wtBRAFT/RAS) cell lines: SKMEL23, KM12, and BT474.

FIG. 5 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for compound BB-01.The ranges of cell lines are as for FIG. 4.

FIG. 6 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for compound BB-02.The ranges of cell lines are as for FIG. 4.

FIG. 7 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for comparisoncompound XX-01. The ranges of cell lines are as for FIG. 4.

FIG. 8 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for comparisoncompound XX-02. The ranges of cell lines are as for FIG. 4.

FIG. 9 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for comparisoncompound XX-03. The ranges of cell lines are as for FIG. 4.

FIG. 10 is a bar graph showing the ratio of the GI₅₀ for each of a rangeof cell lines to the GI₅₀ for the WM266.4 cell line for comparisoncompound XX-04. The ranges of cell lines are as for FIG. 4.

Based on these in vitro data, it would not be expected that the claimedcompounds (especially AA-01 and BB-02) are substantially more effectivethan the comparison compounds (XX-01, XX-02, XX-03, XX-04), in the invivo mutant RAS SW620 xenograft study.

Biological Data—Xenograft Data

Xenograft data for several compounds of the present invention, as wellas several comparison compounds are illustrated in FIGS. 11-19.

FIG. 11 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant RAS cell line SW620, fortreatment with compound AA-01 and for controls.

The data demonstrate that compound AA-01 substantially reduced tumourvolume over the timescale of the study (e.g., a factor of about 3), ascompared to the control, for this mutant RAS colorectal carcinoma cellline.

FIG. 12 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant RAS cell line SW620, fortreatment with compound BB-02 and for controls.

The data demonstrate that compound BB-02 substantially reduced tumourvolume over the timescale of the study (e.g., a factor of about 1.5), ascompared to the control, for this mutant RAS colorectal carcinoma cellline.

FIG. 13 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant BRAF cell line A375, fortreatment with compound AA-01 and for controls.

The data demonstrate that compound AA-01 substantially reduced tumourvolume over the timescale of the study (e.g., a factor of about 2.5), ascompared to the control, for this melanoma cell line.

FIG. 14 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant BRAF cell line A375, fortreatment with compound BB-02 and for controls.

The data demonstrate that compound BB-02 substantially reduced tumourvolume over the timescale of the study (e.g., a factor of about 1.5), ascompared to the control, for this mutant BRAF melanoma cell line.

FIG. 15 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant RAS cell line SW620, fortreatment with comparison compound XX-01 and for controls.

The data demonstrate that comparison compound XX-01 had relativelylittle effect, as compared to the control, for this mutant RAScolorectal carcinoma cell line.

FIG. 16 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant RAS cell line SW620, fortreatment with comparison compound XX-02 and for controls.

The data demonstrate that comparison compound XX-02 had relativelylittle effect, as compared to the control, for this mutant RAScolorectal carcinoma cell line.

FIG. 17 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant RAS cell line SW620, fortreatment with comparison compound XX-03 and for controls.

The data demonstrate that comparison compound XX-03 had relativelylittle effect, as compared to the control, for this mutant RAScolorectal carcinoma cell line.

FIG. 18 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant RAS cell line SW620, fortreatment with comparison compound XX-04 and for controls.

The data demonstrate that comparison compound XX-04 had relativelylittle effect, as compared to the control, for this mutant RAScolorectal carcinoma cell line.

FIG. 19 is a graph of relative tumour volume as a function of time(days) for mouse xenografts of the mutant BRAF cell line A375, fortreatment with comparison compound XX-02 and for controls.

The data demonstrate that comparison compound XX-02 is at least to somedegree effective, as compared to the control, for this mutant BRAFmelanoma cell line.

The ratios of tumour volume (for treatment) to tumour volume (forcontrol) (T/C) for mutant RAS SW620 xenografts are shown in thefollowing table. A lower T/C ratio indicates a higher efficacy. Whereasthe comparison compounds all have T/C ratios which indicate little or noefficacy, the claimed compounds AA-01 and BB-02 both have T/C ratioswhich demonstrate statistically significant efficacy.

TABLE 4 Ratio of: Tumour Volume (Treatment)/ Tumour Volume (Control)Code (mutant RAS SW620 xenograft) AA-01 0.34 BB-02 0.66 XX-01 >1 XX-020.95 XX-03 0.97 XX-04 0.87

The data for the claimed compounds demonstrate that, not only are theclaimed compounds effective as BRAF inhibitors and against mutant BRAFtumours (for example, xenografts of the mutant BRAF melanoma cell lineA375M; FIGS. 13 and 14), but, surprisingly and unexpectedly, the claimedcompounds are also effective against mutant RAS tumours (for example,xenografts of the mutant RAS colorectal carcinoma cell line SW620; FIGS.11 and 12). The surprising and unexpected activity of the claimedcompounds against mutant RAS tumours could not be predicted from theirknown or expected BRAF inhibitory activity.

The data for the comparison compounds demonstrate that, although thecomparison compounds are to some degree effective against mutant BRAFtumours (for example, xenografts of the mutant BRAF melanoma cell lineA375M; FIG. 19), they have relatively little effect against mutant RAStumours (for example, xenografts of the mutant RAS colorectal carcinomacell line SW620; FIGS. 15, 16, 17, and 18).

The foregoing has described the principles, preferred embodiments, andmodes of operation of the present invention. However, the inventionshould not be construed as limited to the particular embodimentsdiscussed. Instead, the above-described embodiments should be regardedas illustrative rather than restrictive, and it should be appreciatedthat variations may be made in those embodiments by workers skilled inthe art without departing from the scope of the present invention.

REFERENCES

A number of patents and publications are cited herein in order to morefully describe and disclose the invention and the state of the art towhich the invention pertains. Full citations for these references areprovided below. Each of these references is incorporated herein byreference in its entirety into the present disclosure, to the sameextent as if each individual reference was specifically and individuallyindicated to be incorporated by reference.

-   Bos, 1989, “ras oncogenes in human cancer: a review”, Cancer Res.,    Vol. 49, pp. 4682-4689.-   Downward, 2003, “Targeting RAS signalling pathways in cancer    therapy”, Nat. Rev. Cancer, Vol. 3, pp. 11-22.-   Garnett et al., 2004, “Guilty as charged: B-RAF is a human    oncogene”, Cancer Cell, Vol. 6, pp. 313-319.-   Gray-Schopfer et al., 2007, “Melanoma biology and new targeted    therapy”, Nature, Vol. 445, pp. 851-857.-   Niculescu-Duvaz et al., 2006, “Imidazo[4,5-b]pyridine-2-one and    oxazolo[4,5-b]pyridine-2-one compounds and analoges thereof as    therapeutic compounds”, international patent application publication    number WO 2006/043090 A1 published 27 Apr. 2006.-   Niculescu-Duvaz et al., 2007, “Imidazo[4,5-b]pyridine-2-one and    oxazolo[4,5-b]pyridine-2-one compounds and analoges thereof as    cancer therapeutic compounds”, international patent application    publication number WO 2007/125330 A1 published 08 Nov. 2007.-   Niculescu-Duvaz et al., 2009, “Aryl-quinolyl compounds and their    use”, international patent application publication number WO    2009/130487 A1 published 29 Oct. 2009.-   Solit et al., 2006, “BRAF mutation predicts sensitivity to MEK    inhibition”, Nature, Vol. 439, pp. 358-362.-   Springer et al., 2009, “Pyrido[2,3-b]pyrazine-8-substituted    compounds and their use”, international patent application    publication number WO 2009/077766 A1 published 25 Jun. 2009.-   Wellbrock et al., 2004, “The RAF proteins take centre stage”, Nature    Reviews Molecular Cell Biology, Vol. 5, pp. 875-885.-   Young et al., 2009, “Ras signaling and therapies”, Adv. Cancer Res.,    Vol. 102, pp. 1-17.

1. A method of inhibiting the progress of, reducing the rate of progressof, alleviating a symptom of, or ameliorating a mutant RAS cancer,comprising administering to a subject in need of treatment atherapeutically-effective amount of a compound selected from compoundsof the following formula, and pharmaceutically acceptable salts thereof:

wherein -J- is independently:

and wherein —R is independently: —H, -Me, —F, —Cl, —Br, or —I; andwherein —R is positioned meta- or para- on the phenyl ring.
 2. A methodaccording to claim 1, wherein the mutant RAS cancer is: mutant RASmelanoma; mutant RAS lung cancer; mutant RAS colorectal cancer; mutantRAS pancreatic cancer; mutant RAS thyroid cancer; mutant RAS seminoma;mutant RAS myelodisplastic syndrome; mutant RAS liver cancer; mutant RASleukemia; mutant RAS sarcoma; mutant RAS lymphoma; mutant RASgastrointestinal cancer; mutant RAS bladder cancer; mutant RAS kidneycancer; mutant RAS ovarian cancer; mutant RAS bile duct cancer; mutantRAS cervix carcinoma; mutant RAS glioma; mutant RAS breast cancer;mutant RAS endometrial cancer; mutant RAS stomach cancer; mutant RAShead and neck cancer; mutant RAS oesophagus carcinoma; or mutant RASprostate cancer.
 3. A method according to claim 1, wherein the mutantRAS cancer is: mutant RAS melanoma; mutant RAS lung cance; mutant RAScolorectal cancer; mutant RAS pancreatic cancer; mutant RAS thyroidcancer; mutant RAS seminoma; mutant RAS myelodisplastic syndrome; mutantRAS liver cancer; mutant RAS leukemia; mutant RAS sarcoma; mutant RASlymphoma; mutant RAS gastrointestinal cancer; mutant RAS bladder cancer;mutant RAS kidney cancer; mutant RAS ovarian cancer; mutant RAS bileduct cancer; or mutant RAS cervix carcinoma.
 4. A method according toclaim 1, wherein the mutant RAS cancer is: mutant RAS melanoma; mutantRAS lung cancer; mutant RAS colorectal cancer; mutant RAS pancreaticcancer; or mutant RAS thyroid cancer.
 5. A method according to claim 4,wherein the compound is selected from compounds of the followingformula, and pharmaceutically acceptable salts thereof:

wherein —R^(A) is independently —H, -Me, —F, —Cl, —Br, or —I; andwherein —R^(A) is positioned meta- or para- on the phenyl ring.
 6. Amethod according to claim 5, wherein —R^(A) is —H.
 7. A method accordingto claim 5, wherein —R^(A) is -Me.
 8. A method according to claim 5,wherein —R^(A) is —F.
 9. A method according to claim 5, wherein —R^(A)is —Cl.
 10. A method according to claim 5, wherein the compound isselected from compounds of the following formula, and pharmaceuticallyacceptable salts thereof:

wherein —R^(A) is independently —H, -Me, —F, —Cl, —Br, or —I.
 11. Amethod according to claim 10, wherein —R^(A) is independently —H, -Me,—F, or —Cl.
 12. A method according to claim 5, wherein the compound isselected from compounds of the following formula, and pharmaceuticallyacceptable salts, hydrates, and solvates thereof:

wherein —R^(A) is independently —H, -Me, —F, —Cl, —Br, or —I.
 13. Amethod according to claim 12, wherein —R^(A) is independently —H, -Me,—F, or —Cl.
 14. A method according to claim 5, wherein the compound isselected from the following compound and pharmaceutically acceptablesalts thereof:


15. A method according to claim 5, wherein the compound is selected fromthe following compound and pharmaceutically acceptable salts thereof:


16. A method according to claim 5, wherein the compound is selected fromthe following compound and pharmaceutically acceptable salts thereof:


17. A method according to claim 5, wherein the compound is selected fromthe following compound and pharmaceutically acceptable salts thereof:


18. A method according to claim 4, wherein the compound is selected fromcompounds of the following formula, and pharmaceutically acceptablesalts thereof:

wherein —R^(B) is independently —H, -Me, —F, —Cl, —Br, or —I; andwherein —R^(B) is positioned meta- or para- on the phenyl ring.
 19. Amethod according to claim 18, wherein —R^(B) is independently —H, -Me,—F, or —Cl.
 20. A method according to claim 18, wherein —R^(B) is —H.21. A method according to claim 18, wherein —R^(B) is -Me.
 22. A methodaccording to claim 18, wherein —R^(B) is —F.
 23. A method according toclaim 18, wherein —R^(B) is —Cl.
 24. A method according to claim 18,wherein the compound is selected from compounds of the followingformula, and pharmaceutically acceptable salts thereof:

wherein —R^(B) is independently —H, -Me, —F, —Cl, —Br, or —I.
 25. Amethod according to claim 24, wherein —R^(B) is independently —H, -Me,—F, or —Cl.
 26. A method according to claim 18, wherein the compound isselected from compounds of the following formula, and pharmaceuticallyacceptable salts thereof:

wherein —R^(B) is independently —H, -Me, —F, —Cl, —Br, or —I.
 27. Amethod according to claim 26, wherein —R^(B) is independently —H, -Me,—F, or —Cl.
 28. A method according to claim 18, wherein the compound isselected from the following compound, and pharmaceutically acceptablesalts thereof:


29. A method according to claim 18, wherein the compound is selectedfrom the following compound, and pharmaceutically acceptable saltsthereof:


30. A method according to claim 18, wherein the compound is selectedfrom the following compound, and pharmaceutically acceptable saltsthereof: